Glaucoma therapeutics and diagnostics

ABSTRACT

Methods and compositions for preventing and treating glaucoma; and glaucoma diagnostics are disclosed.

1. GOVERNMENT SUPPORT

[0001] Work described herein has been supported, in part, by PublicHealth Service Research Grants EY10564, EY08905, EY02477, EY02162,EY08426, P50HG00835 and HG00457. The U.S. Government may therefore havecertain rights in the invention.

2. BACKGROUND OF THE INVENTION

[0002] Glaucoma is an optic nerve disorder characterized by cupping ofthe optic nerve head and loss of peripheral vision. Occasionally thereis also loss of central vision. In the majority of patients, an elevatedintraocular pressure is present and is thought to contribute to theoptic nerve damage. Glaucoma is the second leading cause of blindness indeveloped countries (Leske, M. C. (1983) Am. J. of Epidemiology118:166-191). Its prevalence increases with age and is greater in blackpatients (Leske, M. C. (1983) Am. J. of Epidemiology 118:166-191).Glaucoma affects approximately 2.3 million Americans and blindsapproximately 12,000 of them per year (Tielsch, J. M. (1993) Therapy forglaucoma: costs and consequences. In Transactions of the New OrleansAcademy of Ophthalmologists, S. F. Ball, Franklin, R. M. (Ed.), pp61-68. Kugler, Amsterdam).

[0003] The most prevalent form of glaucoma is primary open angleglaucoma (POAG), a progressive disease of the optic nerve characterizedby degeneration and cupping of the optic nerve, loss of peripheralvisual field, and increased intra-ocular pressure. Evidence indicatesthat POAG is genetically heterogeneous with a complex mode ofinheritance. An early onset form of POAG known as juvenile open angleglaucoma (JOAG) is an autosomal dominant disorder with high penetrance.

[0004] A significant fraction of glaucoma has a genetic basis (Benedict,T. W. G. Abhaundlungen zus dem Gebiete der Augenheilkunde. Breslau: L.Freunde (1842); Stokes, (1940) W. Arch Ophthalmol 24:885-909; Kellerman,L. and A. Posner, (1955) Am. J. Ophthalmol.; 40:681-685; Becker, B., etal., (1960) Am. J. Ophthalmol. 50:557-567; Francois, J., et. al., (1966)Am. J. Ophthalmol.; 62:1067-1071; Armaly, M. F. (1967) Arch Ophthalmol;78:35-43; Davies, T. G. (1968) Br. J Ophthalmol.:52:31-39; Jay, B.,Paterson, G. (1970) Trans. Ophthalmol. Soc. U.K; 90:161-171; Paterson,G. (1970) Trans. Ophthalmol. Soc. U.K; 90:515-525; Miller, S. J. H.(1978) Trans. Ophthalmol. Soc. U.K. 98:290-292), which allows geneticmethods to be used to investigate the pathophysiological mechanisms ofthe disease at the molecular level. The chromosomal locations of genescausing three genetically distinct types of primary open angle glaucomahave been identified (Sheffield, V., et al. (1993) Nature Genetics4:47-50; Sunden, S. L. F., et al. (1996) 6:862-869; Richards, J. E., etal. (1994) Am. J. Hum. Genet.:54:62-70; Wiggs, J. L., et al. (1994)Genomics; 21:299-303; Stoilova, D., et al. (1996) Genomics 36:142-150;Wirtz, M. K., et al. (1997) Am. J. Hum. Genet. 60:296-304).

[0005] Therapeutics, which modulate (agonize or antagonize) genes(wild-type or mutant) involved in glaucoma, would be useful for theprevention and treatment of glaucoma. In addition, the detection ofmutations in genes that correlate with the existence or a predispositionto the development of glaucoma can provide useful diagnostics.

3. SUMMARY OF THE INVENTION

[0006] In one aspect, the invention features isolated GLC1A nucleic acidmolecules. The disclosed molecules can be non-coding, (e.g. probe,antisense or ribozyme molecules) or can encode a functional polypeptide(e.g. a polypeptide which specifically modulates, e.g., by acting aseither an agonist or antagonist, at least one bioactivity of a myocilinpolypeptide).

[0007] In further embodiments, the nucleic acid molecule is a GLC1Anucleic acid that is at least 70%, preferably 80%, more preferably 85%,and even more preferably at least 95% homologous in sequence to thenucleic acids shown as SEQ ID No. 7 or 9 or to the complement thereof.In another embodiment, the nucleic acid molecule encodes a polypeptidethat is at least 92% and more preferably at least 95% similar insequence to the polypeptide shown in SEQ ID No: 8 or 10.

[0008] The invention also provides probes and primers comprisingsubstantially purified oligonucleotides, which correspond to a region ofnucleotide sequence which hybridizes to at least about 6 consecutivenucleotides of the sequences set forth as SEQ ID Nos: 1, 2, 3, 4, 5 or 6or complements of the sequences set forth as SEQ ID Nos: 1, 2, 3, 4, 5or 6 or naturally occurring mutants thereof In preferred embodiments,the probe/primer further includes a label group attached thereto, whichis capable of being detected.

[0009] For expression, the subject GLC1A nucleic acids can include atranscriptional regulatory sequence, e.g. at least one of atranscriptional promoter (e.g., for constitutive expression or inducibleexpression) or transcriptional enhancer or suppressor sequence, whichregulatory sequence is operably linked to the GLC1A gene sequence. Suchregulatory sequences in conjunction with a GLC1A nucleic acid moleculecan provide a usefull vector for gene expression. This invention alsodescribes host cells transfected with said expression vector whetherprokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo(e.g. transgenic) methods for producing GLC1A proteins by employing saidexpression vectors.

[0010] In another aspect, the invention features isolated myocilinpolypeptides, preferably substantially pure preparations, e.g. of plasmapurified or recombinantly produced myocilin polypeptides. In oneembodiment, the polypeptide is identical to or similar to a myocilinprotein represented in SEQ ID No: 8 or 10. Related members of thevertebrate and particularly the mammalian myocilin family are alsowithin the scope of the invention. Preferably, a myocilin polypeptidehas an amino acid sequence at least about 92% homologous and preferablyat least about 95%, 96%, 97%, 98% or 99% homologous to the polypeptiderepresented in SEQ ID No: 8 or 10. In a preferred embodiment, themyocilin polypeptide is encoded by a nucleic acid which hybridizes witha nucleic acid sequence represented in one of SEQ ID No: 7 or 9. Thesubject myocilin proteins also include modified proteins, which areresistant to post-translational modification, as for example, due tomutations which alter modification sites (such as tyrosine, threonine,serine or aspargine residues), or which prevent glycosylation of theprotein, or which prevent interaction of the protein with intracellularproteins involved in signal transduction.

[0011] The myocilin polypeptide can comprise a full length protein, suchas represented in SEQ ID No: 8 or 10, or it can comprise a fragmentcorresponding to one or more particular motifs/domains, or to arbitrarysizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250,275,300, 325, 350, 375, 400, 425, 450, 460, 470, 475, 480, 485, or 490 aminoacids in length.

[0012] Another aspect of the invention features chimeric molecules (e.g.fusion proteins) comprised of a myocilin protein. For instance, themyocilin protein can be provided as a recombinant fusion protein whichincludes a second polypeptide portion, e.g., a second polypeptide havingan amino acid sequence unrelated (heterologous) to the myocilinpolypeptide (e.g. the second polypeptide portion isglutathione-S-transferase, an enzymatic activity such as alkalinephosphatase or an epitope tag).

[0013] Yet another aspect of the present invention concerns an immunogencomprising a myocilin polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for amyocilin polypeptide; e.g. a humoral response, an antibody responseand/or cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g. a unique determinant, from theprotein represented in SEQ ID Nos: 8 or 10.

[0014] A still further aspect of the present invention featuresantibodies and antibody preparations specifically reactive with anepitope of the myocilin protein. In preferred embodiments the antibodyspecifically binds to at least one epitope represented in SEQ ID Nos: 8or 10.

[0015] The invention also features transgenic non-human animals whichinclude (and preferably express) a heterologous form of a GLC1A genedescribed herein, or which misexpress an endogenous GLC1A gene (e.g., ananimal in which expression of one or more of the subject GLC1A proteinsis disrupted). Such a transgenic animal can serve as an animal model forstudying cellular and tissue disorders comprising mutated ormis-expressed GLC1A alleles or for use in drug screening. Alternatively,such a transgenic animal can be useful for expressing recombinantmyocilin polypeptides.

[0016] In yet another aspect, the invention provides assays, e.g., forscreening test compounds to identify inhibitors, or alternatively,potentiators, of an interaction between a myocilin protein and, forexample, a virus, an extracellular ligand of the myocilin protein, or anintracellular protein which binds to the myocilin protein.

[0017] A further aspect of the present invention provides a method ofdetermining if a subject is at risk for glaucoma or another disorderresulting from a mutant GLC1A gene. The method includes detecting, in atissue of the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding amyocilin protein, (e.g., a gene represented in one of SEQ ID Nos: 7 or9, or a homolog thereof or a mutation of a GLC1A intronic sequence, e.g.as represented in SEQ ID Nos. 1-6); or (ii) the mis-expression of aGLC1A gene. In preferred embodiments, detecting the genetic lesionincludes ascertaining the existence of at least one of: a deletion ofone or more nucleotides from a GLC1A gene; an addition of one or morenucleotides to the gene, a substitution of one or more nucleotides ofthe gene, a gross chromosomal rearrangement of the gene; an alterationin the level of a messenger RNA transcript of the gene (e.g., due to apromoter mutation); the presence of a non-wild type splicing pattern ofa messenger RNA transcript of the gene; a non-wild type level of theprotein; and/or an aberrant level of soluble myocilin protein.

[0018] For example, detecting the genetic lesion can include (i)providing a probe/primer comprised of an oligonucleotide whichhybridizes to a sense or antisense sequence of a GLC1A gene or naturallyoccurring mutants thereof, or intronic flanking sequences naturallyassociated with the GLC1A gene; (ii) contacting the probe/primer to anappropriate nucleic acid containing sample; and (iii) detecting, byhybridization of the probe/primer to the nucleic acid, the presence orabsence of the genetic lesion; e.g. wherein detecting the lesioncomprises utilizing the probe/primer to determine the nucleotidesequence of the GLC1A gene and, optionally, of the flanking nucleic acidsequences. For instance, the primer can be employed in a polymerasechain reaction (PCR) or in a ligation chain reaction (LCR). In alternateembodiments, the level of a GLC1A protein is detected in an immunoassayusing an antibody which is specifically immunoreactive with the myocilinprotein.

[0019] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

3. BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is an alignment of human and mouse GLC1A gene sequences.The three exons of the human and mouse GLC1A genes and flankingsequences are aligned in panels A, B and C. These sequences are notcontinuous. Exon sequences are reported in capital letters whileflanking sequences are in lower-case letters. Nucleotides conservedbetween mouse and human are indicated by a closed circle. In panel 1A,exon 1 and flanking promoter and intron 1 sequences are shown. A subsetof putative promoter and enhancer elements are underlined and labeled.GRE half-sites are indicated by “GR”. A (CA) repeat polymorphism in the5′ flanking region of the human GLC1A gene is also underlined andlabeled “(CA) repeat polymorphism”. In panel 1B, exon 2 and flankingintron 1 and intron 2 sequences are shown. In panel 1C, exon 3 andflanking intron 2 and downstream sequences are shown. Polyadenylationsignal sequences are underlined and labeled “poly-A”. A (CA) repeatpolymorphism downstream of the human GLC1A gene is also underlined andlabeled “(CA) repeat polymorphism”.

[0021]FIG. 2 is a schematic representation of putative motifs that areconserved between human and mouse myocilin proteins.

[0022]FIG. 3 is an alignment of the proteins predicted by the mouse andhuman GLC1A genes. Amino acids conserved between mouse and human areindicated by a closed circle. The location of disease-causing mutationspreviously identified in the human GLC1A gene are indicated. For eachmissense mutation, the mutant residue is shown directly above thewild-type amino acid. The location of a nonsense mutation is indicatedby a I and the location of an insertion mutation is indicated by a “2”.

4. DETAILED DESCRIPTION OF THE INVENTION

[0023] 4.1. General

[0024] As reported herein, a genetic locus associated with JOAG wasidentified on chromosome 1q21-q31 by genetic linkage analysis. Observedrecombinations between the glaucoma phenotype and highly polymorphicgenetic markers in two large JOAG kindreds allowed the intervalcontaining GLC1A gene to be narrowed to a 3 cM region of chromosome 1qbetween markers D1S3665 and D1S3664. Further evaluation of markerhaplotypes revealed that each of three pairs of glaucoma families sharedalleles of the same eight contiguous markers suggesting that the GLC1Agene lies within a narrower interval defined by D1S1619 and D1S3664.

[0025] Several genes mapping to the GLC1A region of chromosome I wereconsidered as candidates for the disease-causing gene. Three genes (LAMC1 (H. C. Watkins et. al., (1993) Hum. Mol. Genet. 2: 1084), NPRI (D. G.Lowe et al., (1990) Genomics 8:304), and CNR2 (S. Munro et al., (1993)Nature 365:61), were excluded from the candidate region by geneticlinkage analysis using intragenic polymorphic markers. Five additionalcandidate genes were determined to lie within the observed recombinantinterval by YAC STS content mapping: selectin E (M. P. Bevilacqua etal., (1989) Science 243:1160) (GenBank accession no. M24736); selectin L(T. F. Tedder et al., (1989) J. Exp. Med 170:123) (GenBank accession no.M25280); TXGP-1 (S. Miura et al., (1991) Mol. Cell Biol 11:1313)(GenBank accession no. MD90224; APT1LG1 (T. Takahashi et al., (1994)Int. Immunol. 6, 1567); and TIGR (Trabecular meshwork InducedGlucocorticoid Response Protein) (J. R. Polansky et al., (1989) Prog.Clin. Biol. Res 12:113; J. Escribano et al., (1995) J. Biochem. 118:921;International Patent Application Publication No. WO 96/14411) (GenBankaccession nos. R9549 1, R95447, R95443, R47209). However, two of thesegenes (selectin E, and selectin L) were found to lie outside of theshared haplotype interval with this approach. The remaining genes(APT1LG1, TXGP-1, and TIGR) were found to map within the narrowest JOAGinterval by both YAC STS content and radiation hybrid mapping.

[0026] Two of these genes (APT1LG1 and TIGR) were screened for mutationsin families with JOAG. Primers were selected from the available sequence(T. Takahashi et al., (1994) Int. Immunol. 6, 1567, J. Escribano et al.,(1995) J. Biochem. 118:921; International Patent Application PublicationNo. WO 96/14411) (GenBank accession nos. R95491, R95447, R95443, R47209)and overlapping PCR amplification products were evaluated by singlestrand conformation polymorphism analysis (B. J. Bassam et al., (1991)Anal. Biochem. 196: 80) and direct DNA sequencing. Although the completecDNA sequence of the APT1LG1 and TIGR genes have been published, thepresence of intervening sequences permitted only 85-90% of their codingsequences to be screened in genomic DNA. Eight unrelated JOAG patientswere screened with the APT1LG1 assay but no sequence variants wereidentified.

[0027] The TIGR gene assay was initially used to screen affected membersof four different I q-linked glaucoma families, and affected members offour smaller families implicated by haplotypic data. Amino-acid-alteringmutations were detected in four of eight families. A tyrosine tohistidine mutation in codon 437 was detected in all 22 affected membersof the original family (V. C. Sheffield et al., (1993) Nature Genet.4:47 ) linked to 1q. A glycine to valine mutation in codon 364 wasdetected in two families including one previously unreported adult-onsetopen angle glaucoma family with 15 affected members. A nonsense mutation(glutamine to stop) at codon 368 was detected in two families. Thelatter mutation would be expected to result in a truncation of the geneproduct.

[0028] The prevalence of mutations in the two PCR amplimers thatharbored these three changes was then estimated by screening fourdifferent populations: glaucoma patients with a family history of thedisease; unselected primary open angle glaucoma probands seen in asingle clinic; the general population (approximated by patients withheritable retinal disease and spouses from families who participated inprior linkage studies); and, unrelated volunteers over the age of 40with normal intraocular pressures and no personal or family history ofglaucoma. PCR products determined to contain a sequence variation bySSCP were sequenced and compared to sequence generated from anunaffected individual as well as the normal chromosome in each affectedindividual. Overall, missense or nonsense mutations were found in about3-5% of unrelated glaucoma patients and in about 0.2% of controls. AChi-square test revealed this difference to be significant (p<0.001).

[0029] In a subsequent study, SSCP screening followed by sequencing ofDNA from 1312 unrelated individuals revealed a total of 33 GLC1Asequence changes. Sequencing of the entire GLC1A coding region amplifiedfrom the probands of three families with 1q-linked glaucoma, but withoutSSCP shifts revealed three additional sequence changes. Sixteen of these36 sequence variations (Table 1) met the following criteria for a“probable” disease causing mutation: 1) presence in one or more glaucomapatients; 2) alteration of the predicted amino acid sequence; 3)presence in less than 1% of the general population; 4) absence in the 91normal volunteers. These sixteen mutations were found in 34 of the 716glaucoma probands (4.7%). Ten sequence changes failed to alter thepredicted amino acid sequence of GLC1A and are therefore likely to benon-disease-causing polymorphisms (Table 3). Nine sequence changesaltered the predicted amino acid sequence of GLC1A (eight) or the 5′flanking region (one) but were judged likely to be non-disease-causingpolymorphisms (Table 2) for one of the following reasons. they werepresent in more than 1% of the general population (three), they werefound only in the normal or general population (five), or they werefound in the same allele as a more likely disease-causing mutation(one). TABLE 1 Probable Mutations  1) GLN19HIS  2) ARG82CYS  3)TRP286ARG  4) THR293LYS  5) PRO361SER  6) GLY364VAL  7) GLN368STOP  8)THR377MET  9) ASP380GLY 10) 396INS397 11) ARG422HIS 12) TYR437HIS 13)ALA445VAL 14) ARG470CYS 15) ILE477ASN 16) LYS500ARG

[0030] TABLE 2 Probable Polymorphism 1) GLU352LYS 2) CYS9SER 3) ASN73SER4) ARG76LYS 5) LYS398ARG 6) ARG422CYS 7) SER425PRO 8) TYR473CYS 9)VAL495ILE7

[0031] TABLE 3 Third Nucleotide (Wobble) Polymorphisms  1) PRO13PRO  2)GLY122GLY  3) LEU159LEU  4) LYS2166LYS  5) THR285THR  6) THR325THR  7)VAL329VAL  8) TYR347TYR  9) GLU396GLU 10) VAL439VAL

[0032] Bacterial artificial chromosomes (BACs) containing the human GLC1A gene and its mouse orthologue were subcloned and sequenced to revealthe genomic structure of the genes. Both the human and mouse GLC1A genesare composed of three exons. Human exon 1 (including the 5′ promoterregion of exon 1, base pairs 1-1905; exon 1, base pairs 1906-2509; andthe 5′ end of intron 1, base pairs 2510-2800) is set forth as SEQ IDNo: 1. Human exon 2 (including the 3′ end of intron 1, base pairs 1-193;exon 2, base pairs 194-319; and the 5′ end of intron 2, base pairs320-680) is set forth as SEQ ID No:2. Human exon 3 (including the 3′ endof intron 2, base pairs 1-427; exon 3, base pairs 428-1212; and the 3′UTR, base pairs 1213-2000) is set forth as SEQ ID No:3. Mouse exon 1(including the 5′ promoter region of exon 1; base pairs 1-1947; exon 1,base pairs 1948-2509; and the 5′ end of intron 1, base pairs 2510-2800)is set forth as SEQ ID No:4. Mouse exon 2 (including the 3′ end ofintron 1, base pairs 1-193; exon 2, base pairs 194-319; and the 5′ endof intron 2, base pairs 320-680) is set forth as SEQ ID No: 5 and mouseexon 3 (including the 3′ end of intron 2, base pairs 1-427; exon 3, basepairs 428-1212 and the 3′ UTR, base pairs 1213-1456) is set forth as SEQID No:6. Exons two and three are 126 base pairs and 782 base pairs longin both genes, while exon one is 604 base pairs in the human gene and562 base pairs in the mouse gene. Exon-intron borders are completelyconserved between mouse and human. The human coding GLC1A nucleotidesequence is comprised of 1512 nucleotides (SEQ ID No: 7) and encodes a504 amino acid myocilin protein (SEQ ID NO. 8) having a molecular weightof about 57 kDa. The mouse coding GLC1A nucleotide sequence is comprisedof 1470 nucleotides (SEQ ID No: 9) and encodes a 490 amino acid myocilinprotein (SEQ ID No: 10) having a molecular weight of about 55 kDa. Thehuman and mouse coding sequences are 83% identical at the nucleotidelevel and predict proteins that are 82% identical at the amino acidlevel.

[0033] Many putative transcription regulatory sequences were identifiedin the upstream region of the GLC1A genes (Table 4). Threepoly-adenylation sites were located in the 3′ UTR of the human gene atpositions 1714, 1864 and 2006 base pairs following the putative startcodon. Additionally the human GLC1A gene was found to be closely flankedby two CA simple tandem repeat polymorphisms (STRPs) that proved to beuseful genetic markers for tracing the segregation of the gene withinfamilies. TABLE 4 Putative GLCIA promoter and enhancer elements Humanand Mouse Human only Mouse only AP-1 AFP1 DTF-1 AP-2 CF2-II GATA-2 AP-3CP2 Hb AR DBP Lva c-ETS Elk-1 Lvb-binding factor c-Myc G6 Factor MAFC/EBP HNF-1 MAZ CAC-binding protein HOX-D8 muEBP-C2 Dr HOX-D9 NF-E2 EnHOX-10 PTFI-beta F2F IRF TF3-s GATA-1 LyF-1 USF GFII MBF-1 GR MCBFHiNF-A Myogenin HNF-3 NF-InsE MBF-1 TCF-2alpha MEP-1 TDEF NF-1 TGT3NF-GMb TII N-Oct-3 UBP-1 Oct WT-1 PEA3 Pit-1a PPAR PR PU.1 PuF Sp1 SRYTCF-1A TFIIB TFIIE TFIIF TMF YY1 Zeste

[0034] The human GLC1A gene has been placed on the chromosome I physicalmap between four flanking genes (SELL, SELE, GLC1A, APT1LG1, AT3). Themouse homologs of these flanking genes are present in the same order onthe mouse chromosome 1, suggesting that the mouse GLC1A gene is locatedin this syntenic region between the mouse homologues of SELE andAPT1LG1.

[0035] The expression of human GLC1A was examined by Northern blotanalysis of RNA from adult tissues. High levels of expression of the 2.3kb mRNA was found in a wide range of tissues including: heart, skeletalmuscle, stomach, thyroid, trachea, bone marrow, thymus, prostate, smallintestine and colon. Less abundant GLC1A expression was observed inlung, pancreas, testis, ovary, spinal cord, lymph node and adrenalgland. GLC1A transcripts were not detected in brain, placenta, liver,kidney, spleen or leukocytes. A similar expression pattern was observedin the mouse. To test the possibility that certain regions of the brainwere under represented in poly-A selected mRNA of total brain tissue, aNorthern blot prepared with RNA from several different regions of thebrain were hybridized using a GLC1A probe. Hybridization was observed inthe spinal cord, but not in the cerebellum, cerebral cortex, medulla,occipital lobe, frontal lobe, temporal lobe, or putamen.

[0036]FIG. 2 illustrates protein motifs that are present in both humanand mouse GLC1A proteins. Both the GLC1A nucleic acid sequence andencoded myocilin amino acid sequence show homology to nonmuscle myosinin the N-terminal region and to olfactomedin in the C-terminal region.In addition, both human and mouse GLC1A proteins contain a leucinezipper domain similar to that seen in kinectin and other cytoskeletalproteins in the myosin-like domain (spanning amino acids 71-152). Thismotif consists of two subregions spanning amino acids 71-85 and 103-152in which leucine residues appear three to eight times at every seventhposition. Both the human and the mouse GLC1A nucleic acids include 10putative phosphorylation sites and 4 putative glycosylation sites. Inaddition to these functional domains, a hydrophobic domain appears atthe N-terminus of the myocilin protein and includes a sequenceresembling a signal peptide in which the alanine residue at position 18may be a possible cleavage site.

[0037] Further analysis reveals a hydrophobic region between amino acids17-37 and 426-44. However, the length and degree of hydrophobicity ofthese domains suggests that they are not membrane spanning. Thecarboxy-terminal three amino acids of human GLC1A protein are serine,lysine and methionine. This sequence has been shown to function as aperoxisome targeting sequence in other proteins (Subramani, S (1993)Ann. Rev. of Cell Bio. 9:445-478). However, no such putative targetingsequence is present in the mouse protein. Western blot analysis of humanGLC1A protein reveals bands at 57 and 59 kD, confirming the predictedprotein size and providing evidence that the protein may beglycosylated. These findings suggest that myocilin is a novelcytoskeletal protein involved in the development of neuroepithelium,such as photoreceptor cells.

[0038]FIG. 3 shows an alignment of the predicted amino acid sequence forthe mouse and human GLC1A genes and indicates the position of sixteenmutations with respect to the mouse and human GLC1A protein seqeuences.Fourteen of these mutations are missense mutations that result in singleamino acid substitutions. Twelve of these occur at amino acids that areconserved between human and mouse while two occur at amino acids thatare not conserved. The two remaining mutations include an insertion thatdisrupts two conserved amino acids and a nonsense mutation that resultsin the truncation of the terminal 136 amino acids of the GLC1A proteinand the loss of 121 conserved residues. Thus, the percentage of diseasecausing mutations found in amino acids conserved between mouse and human(88%) is not significantly different from the overall proteinconservation across species (82%).

[0039] Importantly, the GLC1A nucleic acid sequence differssubstantially from the TIGR gene sequence reported in InternationalPatent Application No. WO 96/14411 (GenBank accession nos. R95491,R95447, R95443 and R947209). In fact, as reported, the TIGR genesequence does not encode a functional protein.

[0040] A summary of the differences between the GLC1A gene disclosedherein, and the TIGR gene are presented in Table 5. TABLE 5 DifferencesBetween GLC1A and TIGR Gene Sequences 1. The “C” at bp #331 of the GLC1ADNA coding sequence is not present in the TIGR sequence. 2. The 29 bps“AGGGGCTGCAGAGGGAGCTGGGCACCCTG” (SEQ ID NO. 11) at bp #344-372 of theGLC1A DNA coding sequence are not included in the TIGR sequence. Errors1 and 2 cause the TIGR sequence to wrongly predict 4 amino acids andexclude 10 amino acids from the protein sequence. 3. The “C” at bp #559of the GLC1A DNA coding sequence is not present in the TIGR sequence. 4.A “T” is wrongly inserted between bp #560 and #561 of the GLC1A DNAcoding sequence in the TIGR sequence. Errors 3 and 4 cause the TIGRsequence to incorrectly predict a serine amino acid at residue #187instead of a glutamine. 5. The 9 bps “CTCAGGAGT” present at bps 706-714of the GLC1A DNA coding sequence are wrongly duplicated and insertedbetween bp 714 and 715 in the TIGR sequence. Consequently, the TIGR DNAsequence incorrectly predicts that 3 amino acids are inserted into theGLC1A protein sequence. 6. A “T” is incorrectly inserted between bp #841and #842 of the GLC1A DNA coding sequence in the TIGR sequence. 7. The“G” at bp #891 of the GLC1A DNA coding sequence is not present in theTIGR sequence. Errors 6 and 7 cause 17 amino acids predicted by theGLC1A DNA coding sequence to be out of frame in the TIGR sequence. 8. A“G” at bp #979 of the GLC1A DNA coding sequence is replaced with a “C”in the TIGR sequence. 9. A “C” at bp #980 of the GLC1A DNA codingsequence is replaced with a “G” in the TIGR sequence. Errors 8. and 9.cause the TIGR sequence to wrongly predict an arginine amino acid atresidue #327 instead of an alanine.

[0041] The above 9 errors in the TIGR GLC1A sequence result in 45nucleotide differences that cause 42 incorrect amino acid predictions.Therefore the human TIGR amino acid sequence is only about 91.67%identical to the human myocilin protein sequence and the human TIGR genesequence is only about 97% identical to the human GLC1A sequence.

[0042] The identification of this disease gene increases theunderstanding of the pathophysiology of glaucoma, which in turnfacilitates the development of assays for identifying molecules thatmodulate (e.g. agonize or antagonize) the bioactivity of a functional ormutant TIGR gene or protein. A therapeutically effective amount of thesemolecules can be administered to a subject with glaucoma or at risk fordeveloping glaucoma to prevent or reduce the severity of the condition.

[0043] In addition, the establishment of the disease-causing nature ofeach GLC1A sequence variant and the associated penetrance and age ofonset, as set forth herein, enables a clinician to provide patients, whoharbor a particular sequence change, with useful information regardingtheir risk of developing glaucoma.

[0044] 4.2 Definitions

[0045] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0046] The term “agonist”, as used herein, is meant to refer to an agent(e.g., a myocilin therapeutic) that directly or indirectly enhances,supplements or potentiates a wildtype or mutant myocilin bioactivity.

[0047] The term “antagonist”, as used herein, is meant to refer to anagent (e.g. a myocilin therapeutic) that directly or indirectlyprevents, minimizes or suppresses a wildtype or mutant myocilinbioactivity.

[0048] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0049] A “chimeric protein” or “fusion protein” is a fusion of a firstamino acid sequence encoding one of the subject polypeptides with asecond amino acid sequence defining a domain (e.g. polypeptide portion)foreign to and not substantially homologous with any domain of one ofthe proteins. A chimeric protein may present a foreign domain which isfound (albeit in a different protein) in an organism which alsoexpresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula X-myocilin-Y, wherein myocilin represents at least aportion of the protein which is derived from one of the myocilinproteins, and X and Y are independently absent or represent amino acidsequences which are not related to one of the myocilin sequences in anorganism, including naturally occurring mutants.

[0050] “Complementary” sequences as used herein refer to sequences whichhave sufficient complementarity to be able to hybridize, forming astable duplex.

[0051] A “delivery complex” shall mean a targeting means (e.g. amolecule that results in higher affinity binding of a gene, protein,polypeptide or peptide to a target cell surface and/or increasedcellular uptake by a target cell). Examples of targeting means include:sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome orliposome), viruses (e.g. adenovirus, adeno-associated virus, andretrovirus) or target cell specific binding agents (e.g. ligandsrecognized by target cell specific receptors). Preferred complexes aresufficiently stable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex is cleavableunder appropriate conditions within the cell so that the gene, protein,polypeptide or peptide is released in a functional form.

[0052] As is well known, genes for a particular polypeptide may exist insingle or multiple copies within the genome of an individual. Suchduplicate genes may be identical or may have certain modifications,including nucleotide substitutions, additions or deletions, which allstill code for polypeptides having substantially the same activity. Theterm “DNA sequence encoding a myocilin polypeptide” may thus refer toone or more genes within a particular individual. Moreover, certaindifferences in nucleotide sequences may exist between individualorganisms, which are called alleles. Such allelic differences may or maynot result in differences in amino acid sequence of the encodedpolypeptide yet still encode a protein with the same biologicalactivity.

[0053] As used herein, the term “gene” or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame encoding one ofthe polypeptides of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding a myocilin polypeptide and comprising GLC1A-encoding exonsequences, though it may optionally include intron sequences which areeither derived from a chromosomal GLC1A gene or from an unrelatedchromosomal gene. Exemplary recombinant genes encoding the subjectmyocilin polypeptides are represented in SEQ ID NO 7 and 9. The term“intron” refers to a DNA sequence present in a given GLC1A gene which isnot translated into protein and is generally found between exons.

[0054] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40% identity, though preferably less than 25%identity, with one of the GLC1A sequences of the present invention.

[0055] The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may, forexample, be protein-protein or protein-nucleic acid in nature.

[0056] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject GLC1A polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theGLC1A gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

[0057] The term “modulation” as used herein refers to both upregulation,(i.e., activation or stimulation), for example by agonizing; anddownregulation, (i.e. inhibition or suppression) for example byantagonizing a myocilin bioactivity.

[0058] A “‘myocilin bioactivity’, ‘biological activity’ or ‘activity’”is meant to refer to a cytoskeletal or antigenic function that isdirectly or indirectly preformed by a myocilin polypeptide (whether inits native or denatured conformation), or by any subsequence thereof.Cytoskeletal functions include processes involved with the developmentor structure of ciliated neuroepithelium (e.g. comprising photoreceptorcells). Antigenic functions include possession of an epitope orantigenic site that is capable of cross-reacting with antibodies raisedagainst a naturally occurring or denatured myocilin polypeptide orfragment thereof.

[0059] The “non-human animals” of the invention include mammals such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant GLC1A genes is present and/or expressed or disrupted in sometissues but not others.

[0060] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0061] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0062] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0063] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a myocilin polypeptide is inserted intoa suitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant GLC1A gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native myocilin protein, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the protein.

[0064] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, polypeptides, peptidometics, carbohydrates, lipids orother organic carbon containing or inorganic molecules. Extensivelibraries of chemical or biological (e.g., fungal, bacterial or algalextracts) mixtures are available for screening with the assays of theinvention.

[0065] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeof the invention to hybridize to at least approximately 6, 12, 20, 30,50, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1460, 1470, 1480, 1490 consecutive nucleotides of avertebrate, preferably GLC1A gene, such as a GLC1A sequence designatedin one of SEQ ID Nos: 7 or 9, or a sequence complementary thereto, ornaturally occurring mutants thereof, such that it shows at least 10times more hybridization, preferably at least 50 times morehybridization, and even more preferably at least 100 times morehybridization than it does to a cellular nucleic acid (e.g., mRNA orgenomic DNA) encoding a protein other than a vertebrate GLC1A protein asdefined herein.

[0066] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of one of therecombinant GLC1A genes is under the control of a promoter sequence (orother transcriptional regulatory sequence) which controls the expressionof the recombinant gene in a cell-type in which expression is intended.It will also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofthe naturally-occurring forms of myocilin proteins.

[0067] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a mammalian myocilinpolypeptide or, in the case of anti-sense expression from thetransferred gene, the expression of a naturally-occurring form of themyocilin protein is disrupted.

[0068] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the mammalian myocilin polypeptides, orpending an antisense transcript thereto), which is partly or entirelyheterologous, i.e., foreign, to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

[0069] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the GLC1A proteins, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant GLC1 A geneis silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more GLC1A genes is caused by human intervention, includingboth recombination and antisense techniques.

[0070] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/expression of nucleic acids to which they arelinked. Vectors capable of directing the expression of genes to whichthey are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

[0071] 4.3 Nucleic Acids of the Present Invention

[0072] As described below, one aspect of the invention pertains toisolated nucleic acids comprising nucleotide sequences encoding myocilinpolypeptides, and/or equivalents of such nucleic acids. The termequivalent is understood to include nucleotide sequences encodingfunctionally equivalent myocilin polypeptides or functionally equivalentpeptides having an activity of a vertebrate myocilin protein such asdescribed herein. Equivalent nucleotide sequences will include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, includesequences that differ from the nucleotide sequence of the GLC1A geneshown in SEQ ID Nos: 7 or 9 due to the degeneracy of the genetic code.

[0073] Preferred nucleic acids are vertebrate GLC1A nucleic acids.Particularly preferred vertebrate GLC1A nucleic acids are mammalian.Regardless of species, particularly preferred GLC1A nucleic acids encodepolypeptides that are at least 90% similar to an amino acid sequence ofhuman GLC1A. Preferred nucleic acids encode a GLC1A polypeptidecomprising an amino acid sequence at least 90% homologous and morepreferably 94% homologous with an amino acid sequence of a vertebrateGLC1A, e.g., such as a sequence shown in one of SEQ ID Nos: 8 or 10.Nucleic acids which encode polypeptides at least about 95%, and evenmore preferably at least about 98-99% similarity with an amino acidsequence represented in SEQ ID Nos.: 8 or 10 are also within the scopeof the invention. In a particularly preferred embodiment, the nucleicacid of the present invention encodes an amino acid GLC1A sequence shownin one of SEQ ID No: 8 or 10. In one embodiment, the nucleic acid is acDNA encoding a peptide having at least one bioactivity of the subjectGLC1A polypeptide. Preferably, the nucleic acid includes all or aportion of the nucleotide sequence corresponding to the coding region ofSEQ ID Nos: 1-7 or 9.

[0074] Still other preferred nucleic acids of the present inventionencode a GLC1A polypeptide which includes a polypeptide sequencecorresponding to all or a portion of amino acid residues of SEQ ID Nos:8 or 10, e.g., at least 2, 5, 10, 25, 50, 100, 150 or 200 amino acidresidues of that region. For example, preferred nucleic acid moleculesfor use as probes/primer or antisense molecules (i.e. noncoding nucleicacid molecules) can comprise at least about 6, 12, 20, 30, 50, 100, 125,150 or 200 base pairs in length, whereas coding nucleic acid moleculescan comprise about 200, 250, 300, 350, 400, 410, 420, 430, 435 or 440base pairs.

[0075] Another aspect of the invention provides a nucleic acid whichhybridizes to a nucleic acid represented by one of SEQ ID Nos: 1-7 or 9.Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0 x sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0× SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0× SC at 50° C. to a high stringency of about 0.2× SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed. In a preferred embodiment,a GLC1A nucleic acid of the present invention will bind to one of SEQ IDNos 1 or 2 under moderately stringent conditions, for example at about2.0× SSC and about 40° C. In a particularly preferred embodiment, aGLC1A nucleic acid of the present invention will bind to one of SEQ IDNos: 1-7 or 9 under high stringency conditions.

[0076] Preferred nucleic acids have a sequence at least about 75%homologous and more preferably 80% and even more preferably at leastabout 85% homologous with an amino acid sequence of a mammalian GLC1A,e.g., such as a sequence shown in one of SEQ ID Nos: 8 and 10. Nucleicacids at least about 90%, more preferably about 95%, and most preferablyat least about 98-99% homologous with a nucleic sequence represented inone of SEQ ID Nos: 8 and 10 are of course also within the scope of theinvention. In preferred embodiments, the nucleic acid is a mammalianGLC1A gene and in particularly preferred embodiments, includes all or aportion of the nucleotide sequence corresponding to the coding region ofone of SEQ ID Nos: 1-7 or 9.

[0077] Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID Nos: 1-7 or 9 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a myocilin polypeptide) but differ in sequencefrom the sequence shown in the sequence listing due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of amyocilin polypeptide. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject myocilin polypeptides will exist among mammalians. One skilledin the art will appreciate that these variations in one or morenucleotides (e.g., up to about 3-5% of the nucleotides) of the nucleicacids encoding polypeptides having an activity of a mammalian myocilinpolypeptide may exist among individuals of a given species due tonatural allelic variation.

[0078] As indicated by the examples set out below, myocilinprotein-encoding nucleic acids can be obtained from mRNA present in anyof a number of eukaryotic cells. It should also be possible to obtainnucleic acids encoding mammalian myocilin polypeptides of the presentinvention from genomic DNA from both adults and embryos. For example, agene encoding a myocilin protein can be cloned from either a cDNA or agenomic library in accordance with protocols described herein, as wellas those generally known to persons skilled in the art. Examples oftissues and/or libraries suitable for isolation of the subject nucleicacids include photoreceptor cells of the retina, among others. A cDNAencoding a myocilin protein can be obtained by isolating total mRNA froma cell, e.g. a vertebrate cell, a mammalian cell, or a human cell,including embryonic cells. Double stranded cDNAs can then be preparedfrom the total mRNA, and subsequently inserted into a suitable plasmidor bacteriophage vector using any one of a number of known techniques.The gene encoding a mammalian myocilin protein can also be cloned usingestablished polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. The nucleicacid of the invention can be DNA or RNA. A preferred nucleic acid is acDNA represented by a sequence selected from the group consisting of SEQID Nos: 1 and 2.

[0079] 4.3.1. Vectors.

[0080] This invention also provides expression vectors containing anucleic acid encoding a myocilin polypeptide, operably linked to atleast one transcriptional regulatory sequence. “Operably linked” isintended to mean that the nucleotide sequence is linked to a regulatorysequence in a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the subject mammalian myocilin proteins. Accordingly, theterm “transcriptional regulatory sequence” includes promoters, enhancersand other expression control elements. Such regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). In one embodiment, theexpression vector includes a recombinant gene encoding a peptide havingan agonistic activity of a subject myocilin polypeptide, oralternatively, encoding a peptide which is an antagonistic form of themyocilin protein. Such expression vectors can be used to transfect cellsand thereby produce polypeptides, including fusion proteins, encoded bynucleic acids as described herein. Moreover, the gene constructs of thepresent invention can also be used as a part of a gene therapy protocolto deliver nucleic acids encoding either an agonistic or antagonisticform of one of the subject myocilin proteins. Thus, another aspect ofthe invention features expression vectors for in vivo or in vitrotransfection and expression of a myocilin polypeptide in particular celltypes so as to reconstitute the function of, or alternatively, abrogatethe function of myocilin-induced signaling in a tissue. This could bedesirable, for example, when the naturally-occurring form of the proteinis misexpressed; or to deliver a form of the protein which altersdifferentiation of tissue. Expression vectors may also be employed toinhibit neoplastic transformation.

[0081] In addition to viral transfer methods, such as those illustratedabove, non-viral methods can also be employed to cause expression of asubject myocilin polypeptide in the tissue of an animal. Most nonviralmethods of gene transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inpreferred embodiments, non-viral targeting means of the presentinvention rely on endocytic pathways for the uptake of the subjectmyocilin polypeptide gene by the targeted cell. Exemplary targetingmeans of this type include liposomal derived systems, poly-lysineconjugates, and artificial viral envelopes.

[0082] 4.3.2. Probes and Primers

[0083] Moreover, the nucleotide sequences determined from the cloning ofGLC1A genes from mammalian organisms will further allow for thegeneration of probes and primers designed for use in identifying and/orcloning homologs in other cell types, e.g. from other tissues, as wellas homologs from other mammalian organisms. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast approximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or anti-sense sequence selected fromthe group consisting of SEQ ID Nos: 1-7 or 9, or naturally occurringmutants thereof For instance, primers based on the nucleic acidrepresented in SEQ ID Nos: 1-7 or 9 can be used in PCR reactions toclone homologs. Preferred primer pairs of the invention are set forth asSEQ ID Nos. 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; 22and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and35; 36 and 37; 38 and 39; 40 and 41; 42 and 43; 44 and 45; and 46 and47.

[0084] Likewise, probes based on the subject GLC1A sequences can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto and able to be detected, e.g.the label group can be selected from amongst radioisotopes, fluorescentcompounds, enzymes, and enzyme co-factors, etc.

[0085] As discussed in more detail below, such probes can also be usedas a part of a diagnostic test kit for identifying cells or tissue whichmisexpress a myocilin protein, such as by measuring a level of amyocilin -encoding nucleic acid in a sample of cells from a patient;e.g. detecting GLC1A mRNA levels or determining whether a genomic GLC1Agene has been mutated or deleted. Briefly, nucleotide probes can begenerated from the subject GLC1A genes which facilitate histologicalscreening of intact tissue and tissue samples for the presence (orabsence) of myocilin-encoding transcripts. Similar to the diagnosticuses of anti-myocilin antibodies, the use of probes directed to GLC1Amessages, or to genomic GLC1A sequences, can be used for both predictiveand therapeutic evaluation of subjects. Used in conjunction withimmunoassays as described herein, the oligonucleotide probes can helpfacilitate the determination of the molecular basis for a developmentaldisorder which may involve some abnormality associated with expression(or lack thereof) of a myocilin protein. For instance, variation inpolypeptide synthesis can be differentiated from a mutation in a codingsequence.

[0086] 4.3.3. Antisense, Ribozyme and Triplex Techniques

[0087] One aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g. bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject GLC1A proteins so as to inhibitexpression of that protein, e.g. by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

[0088] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thecell, produces RNA which is complementary to at least a unique portionof the cellular mRNA which encodes a myocilin protein. Alternatively,the antisense construct is an oligonucleotide probe which is generatedex vivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of aGLC1A gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the GLC1A nucleotide sequence of interest, are preferred.

[0089] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to GLC1A mRNA. The antisenseoligonucleotides will bind to the GLC1A mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. A sequence “complementary” to a portion of an RNA, as referredto herein, means a sequence having sufficient complementarity to be ableto hybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

[0090] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. (Wagner, R. 1994. Nature372:333). Therefore, oligonucleotides complementary to either the 5′ or3′ untranslated, non-coding regions of a GLC1A gene could be used in anantisense approach to inhibit translation of endogenous GLC1A mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5′, 3′ or coding regionof GLC1A mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In certain embodiments, theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides, or at least 50 nucleotides.

[0091] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to quantitate the ability of theantisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

[0092] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad Sci.84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0093] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0094] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0095] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0096] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual conformation, the strands run parallel to each other (Gautier etal., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0097] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0098] While antisense nucleotides complementary to the GLC1A codingregion sequence could be used, those complementary to the transcribeduntranslated region are most preferred.

[0099] The antisense molecules should be delivered to cells whichexpress the myocilin in vivo. A number of methods have been developedfor delivering antisense DNA or RNA to cells; e.g., antisense moleculescan be injected directly into the tissue site, or modified antisensemolecules, designed to target the desired cells (e.g., antisense linkedto peptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface) can be administeredsystematically.

[0100] However, it is often difficult to achieve intracellularconcentrations of the antisense sufficient to suppress translation ofendogenous mRNAs. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous GLC1A transcripts andthereby prevent translation of the GLC1A mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systematically).

[0101] Ribozyme molecules designed to catalytically cleave GLC1A mRNAtranscripts can also be used to prevent translation of GLC1A mRNA andexpression of myocilin. (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy GLC1A mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.There are hundreds of potential hammerhead ribozyme cleavage siteswithin the nucleotide sequence of human GLC1A cDNA. Preferably theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the GLC1A mRNA; i.e., to increase efficiency andminimize the intracellular accumulation of non-functional mRNAtranscripts.

[0102] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in GLC1A.

[0103] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.) and should be delivered to cells which express the GLC1A in vivoe.g., hypothalamus and/or the choroid plexus. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy endogenous GLC1A messages and inhibit translation. Becauseribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

[0104] Endogenous GLC1A gene expression can also be reduced byinactivating or “knocking out” the GLC1A gene or its promoter usingtargeted homologous recombination. (E.g., see Smithies et al., 1985,Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompsonet al., 1989 Cell 5:313-321; each of which is incorporated by referenceherein in its entirety). For example, a mutant, non-functional GLC1A (ora completely unrelated DNA sequence) flanked by DNA homologous to theendogenous GLC1A gene (either the coding regions or regulatory regionsof the GLC1A gene) can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that expressGLC1A in vivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the GLC1A gene. Suchapproaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive GLC1A (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be adapted foruse in humans provided the recombinant DNA constructs are directlyadministered or targeted to the required site in vivo using appropriateviral vectors, e.g., herpes virus vectors for delivery to brain tissue;e.g., the hypothalamus and/or choroid plexus.

[0105] Alternatively, endogenous GLC1A gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the GLC1A gene (i.e., the GLC1A promoter and/or enhancers) toform triple helical structures that prevent transcription of the GLC1Agene in target cells in the body. (See generally, Helene, C. 1991,Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y.Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

[0106] Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the myocilinproteins, can be used in the manipulation of tissue, e.g. tissuedifferentiation, both in vivo and for ex vivo tissue cultures.

[0107] Furthermore, the anti-sense techniques (e.g. microinjection ofantisense molecules, or transfection with plasmids whose transcripts areantisense with regard to a GLC1A mRNA or gene sequence) can be used toinvestigate role of myocilin in developmental events, as well as thenormal cellular function of myocilin in adult tissue. Such techniquescan be utilized in cell culture, but can also be used in the creation oftransgenic animals, as detailed below.

[0108] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the target gene mRNA, and must include the well knowncatalytic sequence responsible for mRNA cleavage. For this sequence, seeU.S. Pat. No. 5,093,246, which is incorporated by reference herein inits entirety. As such within the scope of the invention are engineeredhammerhead motif ribozyme molecules that specifically and efficientlycatalyze endonucleolytic cleavage of RNA sequences encoding myocilinproteins.

[0109] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the molecule of interest forribozyme cleavage sites which include the following sequences, GUA, GUUand GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

[0110] Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription are preferably single stranded andcomposed of deoxyribonucleotides. The base composition of theseoligonucleotides should promote triple helix formation via Hoogsteenbase pairing rules, which generally require sizable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

[0111] Alternatively, the potential sequences that can be targeted fortriple helix formation may be increased by creating a so called“switchback” nucleic acid molecule. Switchback molecules are synthesizedin an alternating 5′-3′, 3′-5′ manner, such that they base pair withfirst one strand of a duplex and then the other, eliminating thenecessity for a sizable stretch of either purines or pyrimidines to bepresent on one strand of a duplex.

[0112] Antisense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

[0113] Moreover, various well-known modifications to nucleic acidmolecules may be introduced as a means of increasing intracellularstability and half-life. Possible modifications include but are notlimited to the addition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

[0114] 4.4. Polypeptides of the Present Invention

[0115] The present invention also makes available myocilin polypeptides,which are isolated from, or otherwise substantially free of othercellular proteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the myocilinpolypeptide. The term “substantially free of other cellular proteins”(also referred to herein as “contaminating proteins”) or “substantiallypure or purified preparations” are defined as encompassing preparationsof myocilin polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein. By “purified”, it is meant, whenreferring to a peptide or DNA or RNA sequence, that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term “purified” as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above. “Isolated” and “purified” do not encompass eithernatural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but notobtained either as pure (e.g. lacking contaminating proteins, orchromatography reagents such as denaturing agents and polymers, e.g.acrylamide or agarose) substances or solutions. In preferredembodiments, purified GLC1A preparations will lack any contaminatingproteins from the same animal from which myocilin is normally produced,as can be accomplished by recombinant expression of, for example, ahuman myocilin protein in a non-human cell.

[0116] Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length are withinthe scope of the present invention.

[0117] For example, isolated myocilin polypeptides can include all or aportion of an amino acid sequences corresponding to a myocilinpolypeptide represented in SEQ ID Nos: 8 or 10. Isolated peptidylportions of myocilin proteins can be obtained by screening peptidesrecombinantly produced from the corresponding fragment of the nucleicacid encoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, a myocilinpolypeptide of the present invention may be arbitrarily divided intofragments of desired length with no overlap of the fragments, orpreferably divided into overlapping fragments of a desired length. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments which can function as eitheragonists or antagonists of a wild-type (e.g., “authentic”) myocilinprotein.

[0118] Another aspect of the present invention concerns recombinantforms of the myocilin proteins. Recombinant polypeptides preferred bythe present invention, in addition to native myocilin proteins, are atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous with anamino acid sequence represented by SEQ ID Nos: 8 or 10. In a preferredembodiment, a myocilin protein of the present invention is a myocilinprotein. In a particularly preferred embodiment, a myocilin proteincomprises the coding sequence of one of SEQ ID No. 1-7, or 9. Inparticularly preferred embodiments, a myocilin protein has a myocilinbioactivity.

[0119] The present invention further pertains to recombinant forms ofone of the subject myocilin polypeptides which are encoded by genesderived from a mammalian organism, and which have amino acid sequencesevolutionarily related to the myocilin proteins represented in SEQ IDNos: 8 or 10. Such recombinant myocilin polypeptides preferably arecapable of functioning in one of either role of an agonist or antagonistof at least one biological activity of a wild-type (“authentic”)myocilin protein of the appended sequence listing. The term“evolutionarily related to”, with respect to amino acid sequences ofmyocilin proteins, refers to both polypeptides having amino acidsequences which have arisen naturally, and also to mutational variantsof myocilin polypeptides which are derived, for example, bycombinatorial mutagenesis. Such evolutionarily derived myocilinpolypeptides preferred by the present invention have a myocilinbioactivity and are at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% homologous with the amino acid sequence selected from the groupconsisting of SEQ ID Nos: 8 or 10.

[0120] In general, polypeptides referred to herein as having an activityof a myocilin protein (e.g., are “bioactive”) are defined aspolypeptides which include an amino acid sequence corresponding (e.g.,identical or homologous) to all or a portion of the amino acid sequencesof a myocilin protein shown in SEQ ID Nos: 8 or 10 and which mimic orantagonize all or a portion of the biological/biochemical activities ofa naturally occurring myocilin protein. According to the presentinvention, a polypeptide has biological activity if it is a specificagonist or antagonist of a naturally-occurring form of a myocilinprotein.

[0121] The present invention further pertains to methods of producingthe subject myocilin polypeptides. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells may beharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The recombinant myocilin polypeptide can beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for such peptide. In a preferred embodiment, the recombinantmyocilin polypeptide is a fusion protein containing a domain whichfacilitates its purification, such as GST fusion protein or poly(His)fusion protein.

[0122] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject myocilin polypeptides which function in a limited capacity asone of either a myocilin agonist (mimetic) or a myocilin antagonist, inorder to promote or inhibit only a subset of the biological activitiesof the naturally-occurring form of the protein. Thus, specificbiological effects can be elicited by treatment with a homolog oflimited function, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally occurring forms of myocilin proteins.

[0123] Homologs of each of the subject myocilin proteins can begenerated by mutagenesis, such as by discrete point mutation(s), or bytruncation. For instance, mutation can give rise to homologs whichretain substantially the same, or merely a subset, of the biologicalactivity of the myocilin polypeptide from which it was derived.Alternatively, antagonistic forms of the protein can be generated whichare able to inhibit the function of the naturally occurring form of theprotein, such as by competitively binding to a downstream or upstreammember of the biochemical pathway, which includes the myocilin protein.In addition, agonistic forms of the protein may be generated which areconstitutively active. Thus, the human myocilin protein and homologsthereof provided by the subject invention may be either positive ornegative regulators of gene expression.

[0124] The recombinant myocilin polypeptides of the present inventionalso include homologs of the authentic myocilin proteins, such asversions of those protein which are resistant to proteolytic cleavage,as for example, due to mutations which alter ubiquitination or otherenzymatic targeting associated with the protein.

[0125] Myocilin polypeptides may also be chemically modified to createderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of myocilin proteins can beprepared by lining the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

[0126] Modification of the structure of the subject myocilinpolypeptides can be for such purposes as enhancing therapeutic orprophylactic efficacy, stability (e.g., ex vivo shelf life andresistance to proteolytic degradation in vivo), or post-translationalmodifications (e.g., to alter phosphorylation pattern of protein). Suchmodified peptides, when designed to retain at least one activity of thenaturally-occurring form of the protein, or to produce specificantagonists thereof, are considered functional equivalents of themyocilin polypeptides described in more detail herein. Such modifiedpeptides can be produced, for instance, by amino acid substitution,deletion, or addition.

[0127] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are can be divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. In similar fashion, the amino acid repertoire can be groupedas (1) acidic=aspartate, glutamate; (2) basic=lysine, argininehistidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally be groupedseparately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine,tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. (see, for example, Biochemisty, 2nded., Ed. by L. Stryer, WH Freeman and Co.: 1981). Whether a change inthe amino acid sequence of a peptide results in a functional myocilinhomolog (e.g. functional in the sense that the resulting polypeptidemimics or antagonizes the wild-type form) can be readily determined byassessing the ability of the variant peptide to produce a response incells in a fashion similar to the wild-type protein, or competitivelyinhibit such a response. Polypeptides in which more than one replacementhas taken place can readily be tested in the same manner.

[0128] This invention further contemplates a method for generating setsof combinatorial mutants of the subject myocilin proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g. homologs) that are functional in modulating geneexpression. The purpose of screening such combinatorial libraries is togenerate, for example, novel myocilin homologs which can act as eitheragonists or antagonist, or alternatively, possess novel activities alltogether.

[0129] Likewise, myocilin homologs can be generated by the presentcombinatorial approach to selectively inhibit gene expression. Forinstance, mutagenesis can provide myocilin homologs which are able tobind other signal pathway proteins (or DNA) yet prevent propagation ofthe signal, e.g. the homologs can be dominant negative mutants.Moreover, manipulation of certain domains of myocilin by the presentmethod can provide domains more suitable for use in fusion proteins.

[0130] In one embodiment, the variegated library of variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential GLC1A sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display) containing the set ofGLC1A sequences therein.

[0131] There are many ways by which such libraries of potential myocilinhomologs can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then ligated intoan appropriate expression vector. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential myocilin sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierppg. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0132] Likewise, a library of coding sequence fragments can be providedfor a GLC1A clone in order to generate a variegated population ofmyocilin fragments for screening and subsequent selection of bioactivefragments. A variety of techniques are known in the art for generatingsuch libraries, including chemical synthesis. In one embodiment, alibrary of coding sequence fragments can be generated by (i) treating adouble stranded PCR fragment of a GLC1A coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule;(i) denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

[0133] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of GLC1A homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate GLC1A sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 10²⁶ molecules. Combinatorial libraries of this size may betechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-331).

[0134] The invention also provides for reduction of the myocilinproteins to generate mimetics, e.g. peptide or non-peptide agents, whichare able to disrupt binding of a mammalian myocilin polypeptide of thepresent invention with either upstream or downstream components. Thus,such mutagenic techniques as described above are also useful to map thedeterminants of the myocilin proteins which participate inprotein-protein interactions involved in, for example, binding of thesubject myocilin polypeptide to proteins which may function upstream(including both activators and repressors of its activity) or toproteins or nucleic acids which may function downstream of the myocilinpolypeptide, whether they are positively or negatively regulated by it.To illustrate, the critical residues of a subject myocilin polypeptidewhich are involved in molecular recognition of a component upstream ordownstream of myocilin can be determined and used to generatemyocilin-derived peptidomimetics which competitively inhibit binding ofthe authentic myocilin protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of each ofthe subject myocilin proteins which are involved in binding otherextracellular proteins, peptidominetic compounds can be generated whichmimic those residues of the myocilin protein which facilitate theinteraction. Such mimetics may then be used to interfere with the normalfunction of a myocilin protein. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al, in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J. Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

[0135] 4.4.1. Cells Expressing Recombinant Myocilin Polypeptides.

[0136] This invention also pertains to a host cell transfected toexpress a recombinant form of the subject myocilin polypeptides. Thehost cell may be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of myocilin proteins, encoding all ora selected portion of the full-length protein, can be used to produce arecombinant form of a myocilin polypeptide via microbial or eukaryoticcellular processes. Ligating the polynucleotide sequence into a geneconstruct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial) cells, are standard proceduresused in producing other well-known proteins, e.g. MAP kinase, pg. 53,WT1, PTP phosphotases, SRC, and the like. Similar procedures, ormodifications thereof, can be employed to prepare recombinant myocilinpolypeptides by microbial means or tissue-culture technology in accordwith the subject invention.

[0137] The recombinant GLC1A genes can be produced by ligating nucleicacid encoding a myocilin protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject myocilin polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of a myocilin polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

[0138] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a myocilin polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the GLC1A genes represented in SEQ ID Nos:1-7 or 9.

[0139] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (13PV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed, by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0140] In some instances, it may be desirable to express the recombinantmyocilin polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

[0141] When it is desirable to express only a portion of a myocilinprotein, such as a form lacking a portion of the N-terminus, i.e. atruncation mutant which lacks the signal peptide, it may be necessary toadd a start codon (ATG) to the oligonucleotide fragment containing thedesired sequence to be expressed. It is well known in the art that amethionine at the N-terminal position can be enzymatically cleaved bythe use of the enzyme methionine aminopeptidase (MAP). MAP has beencloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757)and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins Miller et al. (1987) PNAS84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing myocilin-derivedpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al., supra).

[0142] In other embodiments transgenic animals, described in more detailbelow could be used to produce recombinant proteins.

[0143] 4.4.2 Fusion Proteins and Immunogens.

[0144] In another embodiment, the coding sequences for the polypeptidecan be incorporated as a part of a fusion gene including a nucleotidesequence encoding a different polypeptide. This type of expressionsystem can be useful under conditions where it is desirable to producean immunogenic fragment of a myocilin protein. For example, the VP6capsid protein of rotavirus can be used as an immunologic carrierprotein for portions of the myocilin polypeptide, either in themonomeric form or in the form of a viral particle. The nucleic acidsequences corresponding to the portion of a subject myocilin protein towhich antibodies are to be raised can be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising myocilin epitopes as part of the virion. Ithas been demonstrated with the use of immunogenic fusion proteinsutilizing the Hepatitis B surface antigen fusion proteins thatrecombinant Hepatitis B virions can be utilized in this role as well.Similarly, chimeric constructs coding for fusion proteins containing aportion of a myocilin protein and the poliovirus capsid protein can becreated to enhance immunogenicity of the set of polypeptide antigens(see, for example, EP Publication No: 0259149; and Evans et al. (1989)Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger etal. (1992) J. Virol. 66:2).

[0145] The Multiple Antigen Peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of a myocilin polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofmyocilin proteins can also be expressed and presented by bacterialcells.

[0146] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the myocilin polypeptides of the present invention.For example, myocilin polypeptides can be generated asglutathione-S-transferase (GST-fusion) proteins. Such GST-fusionproteins can enable easy purification of the myocilin polypeptide, asfor example by the use of glutathione-derivatized matrices (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.(N.Y.: John Wiley & Sons, 1991)).

[0147] In another embodiment, a fusion gene coding for a purificationleader sequence, such as a poly-(His)/enterokinase cleavage sitesequence at the N-terminus of the desired portion of the recombinantprotein, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni2⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified protein (e.g., see Hochuli et al.(1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

[0148] 4.4.3. Antibodies

[0149] Another aspect of the invention pertains to an antibody orbinding fragment thereof, which is specifically reactive with a myocilinprotein. For example, by using immunogens derived from a myocilinprotein, e.g. based on the cDNA sequences, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of thepeptide (e.g., a myocilin polypeptide or an antigenic fragment which iscapable of eliciting an antibody response, or a fusion protein asdescribed above). Techniques for conferring immunogenicity on a proteinor peptide include conjugation to carriers or other techniques wellknown in the art. An immunogenic portion of a myocilin protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofa myocilin protein of a mammal, e.g. antigenic determinants of a proteinrepresented by SEQ ID No: 2 or closely related homologs (e.g. at least92% homologous, and more preferably at least 94% homologous).

[0150] Following immunization of an animal with an antigenic preparationof a myocilin polypeptide, anti-myocilin antisera can be obtained and,if desired, polyclonal anti-myocilin antibodies isolated from the serum.To produce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a myocilinpolypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells.

[0151] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian myocilin polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having affinityfor a myocilin protein conferred by at least one CDR region of theantibody.

[0152] Antibodies which specifically bind myocilin epitopes can also beused in immunohistochemical staining of tissue samples in order toevaluate the abundance and pattern of expression of each of the subjectmyocilin polypeptides. Anti-myocilin antibodies can be useddiagnostically in immuno-precipitation and immuno-blotting to detect andevaluate myocilin protein levels in tissue as part of a clinical testingprocedure. For instance, such measurements can be useful in predictivevaluations of the onset or progression of proliferative disorders.Likewise, the ability to monitor myocilin protein levels in anindividual can allow determination of the efficacy of a given treatmentregimen for an individual afflicted with such a disorder. The level ofmyocilin polypeptides may be measured from cells in bodily fluid, suchas in samples of cerebral spinal fluid or amniotic fluid, or can bemeasured in tissue, such as produced by biopsy. Diagnostic assays usinganti-myocilin antibodies can include, for example, immunoassays designedto aid in early diagnosis of a degenerative disorder, particularly oneswhich are manifest at birth. Diagnostic assays using anti-myocilinpolypeptide antibodies can also include immunoassays designed to aid inearly diagnosis and phenotyping neoplastic or hyperplastic disorders.

[0153] Another application of anti-myocilin antibodies of the presentinvention is in the immunological screening of cDNA librariesconstructed in expression vectors such as gt11, gt18-23, ZAP, and ORF8.Messenger libraries of this type, having coding sequences inserted inthe correct reading frame and orientation, can produce fusion proteins.For instance, gt11 will produce fusion proteins whose amino terminiconsist of β-galactosidase amino acid sequences and whose carboxytermini consist of a foreign polypeptide. Antigenic epitopes of amyocilin protein, e.g. other orthologs of a particular myocilin proteinor other paralogs from the same species, can then be detected withantibodies, as, for example, reacting nitrocellulose filters lifted frominfected plates with anti-myocilin antibodies. Positive phage detectedby this assay can then be isolated from the infected plate. Thus, thepresence of myocilin homologs can be detected and cloned from otheranimals, as can alternate isoforms (including splicing variants) fromhumans.

[0154] 4.5 Transgenic Animals

[0155] The invention further provides for transgenic animals, which canbe used for a variety of purposes, e.g., to identify myocilintherapeutics. Transgenic animals of the invention include non-humananimals containing a heterologous GLC1A gene or fragment thereof underthe control of a GLC1A promoter or under the control of a heterologouspromoter. Accordingly, the transgenic animals of the invention can beanimals expressing a transgene encoding a wild-type myocilin protein orfragment thereof or variants thereof, including mutants and polymorphicvariants thereof Such animals can be used, e.g., to determine the effectof a difference in amino acid sequence of a myocilin protein from thesequence set forth in SEQ ID NOS. 8 or 10, such as a polymorphicdifference. These animals can also be used to determine the effect ofexpression of a myocilin protein in a specific site or for identifyingmyocilin therapeutics or confirming their activity in vivo.

[0156] The transgenic animals can also be animals containing atransgene, such as reporter gene, under the control of a GLC1A promoteror fragment thereof. These animals are useful, e.g., for identifyingdrugs that modulate production of myocilin, such as by modulating GLC1Agene expression. A GLC1A gene promoter can be isolated, e.g., byscreening of a genomic library with a GLC1A cDNA fragment andcharacterized according to methods known in the art. In a preferredembodiment of the present invention, the transgenic animal containingsaid GLC1A reporter gene is used to screen a class of bioactivemolecules known as steroid hormones for their ability to modulate GLC1Aexpression.

[0157] Yet other non-human animals within the scope of the inventioninclude those in which the expression of the endogenous GLC1A gene hasbeen mutated or “knocked out”. A “knock ouf” animal is one carrying ahomozygous or heterozygous deletion of a particular gene or genes. Theseanimals could be used to determine whether the absence of GLC1A willresult in a specific phenotype, in particular whether these mice have orare likely to develop a specific disease, such as high susceptibility toheart disease or cancer. Furthermore these animals are useful in screensfor drugs which alleviate or attenuate the disease condition resultingfrom the mutation of the GLC1A gene as outlined below. These animals arealso useful for determining the effect of a specific amino aciddifference, or allelic variation, in a GLC1A gene. That is, the GLC1Aknock out animals can be crossed with transgenic animals expressing,e.g., a mutated form or allelic variant of GLC1A, thus resulting in ananimal which expresses only the mutated protein and not the wild-typemyocilin protein.

[0158] Methods for obtaining transgenic and knockout non-human animalsare well known in the art. Knock out mice are generated by homologousintegration of a “knock out” construct into a mouse embryonic stem cellchromosome which encodes the gene to be knocked out. In one embodiment,gene targeting, which is a method of using homologous recombination tomodify an animal's genome, can be used to introduce changes intocultured embryonic stem cells. By targeting a GLC1A gene of interest inES cells, these changes can be introduced into the germlines of animalsto generate chimeras. The gene targeting procedure is accomplished byintroducing into tissue culture cells a DNA targeting construct thatincludes a segment homologous to a target GLC1A locus, and which alsoincludes an intended sequence modification to the GLC1A genomic sequence(e.g., insertion, deletion, point mutation). The treated cells are thenscreened for accurate targeting to identify and isolate those which havebeen properly targeted.

[0159] Gene targeting in embryonic stem cells is in fact a schemecontemplated by the present invention as a means for disrupting a GLC1Agene function through the use of a targeting transgene constructdesigned to undergo homologous recombination with one or more GLC1Agenomic sequences. The targeting construct can be arranged so that, uponrecombination with an element of a GLC1A gene, a positive selectionmarker is inserted into (or replaces) coding sequences of the gene. Theinserted sequence functionally disrupts the GLC1A gene, while alsoproviding a positive selection trait. Exemplary GLC1A targetingconstructs are described in more detail below.

[0160] Generally, the embryonic stem cells (ES cells ) used to producethe knockout animals will be of the same species as the knockout animalto be generated. Thus for example, mouse embryonic stem cells willusually be used for generation of knockout mice.

[0161] Embryonic stem cells are generated and maintained using methodswell known to the skilled artisan such as those described by Doetschmanet al. (1985) J. Embryol. Exp. 87:27-45). Any line of ES cells can beused, however, the line chosen is typically selected for the ability ofthe cells to integrate into and become part of the germ line of adeveloping embryo so as to create germ line transmission of the knockoutconstruct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934) Still another preferred ES cell line is the WW6cell line (Ioffe et al. (1995) PNAS 92:7357-7361). The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth by Robertsonin: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al.(1986) Current Topics in Devel. Biol 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]).

[0162] A knock out construct refers to a uniquely configured fragment ofnucleic acid which is introduced into a stem cell line and allowed torecombine with the genome at the chromosomal locus of the gene ofinterest to be mutated. Thus a given knock out construct is specific fora given gene to be targeted for disruption. Nonetheless, many commonelements exist among these constructs and these elements are well knownin the art. A typical knock out construct contains nucleic acidfragments of not less than about 0.5 kb nor more than about 10.0 kb fromboth the 5′ and the 3′ ends of the genomic locus which encodes the geneto be mutated. These two fragments are separated by an interveningfragment of nucleic acid which encodes a positive selectable marker,such as the neomycin resistance gene (neo^(R)). The resulting nucleicacid fragment, consisting of a nucleic acid from the extreme 5′ end ofthe genomic locus linked to a nucleic acid encoding a positiveselectable marker which is in turn linked to a nucleic acid from theextreme 3′ end of the genomic locus of interest, omits most of thecoding sequence for GLC1A or other gene of interest to be knocked out.When the resulting construct recombines homologously with the chromosomeat this locus, it results in the loss of the omitted coding sequence,otherwise known as the structural gene, from the genomic locus. A stemcell in which such a rare homologous recombination event has taken placecan be selected for by virtue of the stable integration into the genomeof the nucleic acid of the gene encoding the positive selectable markerand subsequent selection for cells expressing this marker gene in thepresence of an appropriate drug (neomycin in this example). Variationson this basic technique also exist and are well known in the art. Forexample, a “knock-in” construct refers to the same basic arrangement ofa nucleic acid encoding a 5′ genomic locus fragment linked to nucleicacid encoding a positive selectable marker which in turn is linked to anucleic acid encoding a 3′ genomic locus fragment, but which differs inthat none of the coding sequence is omitted and thus the 5′ and the 3′genomic fragments used were initially contiguous before being disruptedby the introduction of the nucleic acid encoding the positive selectablemarker gene. This “knock-in”type of construct is thus very useful forthe construction of mutant transgenic animals when only a limited regionof the genomic locus of the gene to be mutated, such as a single exon,is available for cloning and genetic manipulation. Alternatively, the“knock-in” construct can be used to specifically eliminate a singlefunctional domain of the targeted gene, resulting in a transgenic animalwhich expresses a polypeptide of the targeted gene which is defective inone function, while retaining the function of other domains of theencoded polypeptide. This type of “knock-in” mutant frequently has thecharacteristic of a so-called “dominant negative” mutant because,especially in the case of proteins which homomultimerize, it canspecifically block the action of (or “poison”) the polypeptide productof the wild-type gene from which it was derived. In a variation of theknock-in technique, a marker gene is integrated at the genomic locus ofinterest such that expression of the marker gene comes under the controlof the transcriptional regulatory elements of the targeted gene. Amarker gene is one that encodes an enzyme whose activity can be detected(e.g., β-galactosidase), the enzyme substrate can be added to the cellsunder suitable conditions, and the enzymatic activity can be analyzed.One skilled in the art will be familiar with other useful markers andthe means for detecting their presence in a given cell. All such markersare contemplated as being included within the scope of the teaching ofthis invention.

[0163] As mentioned above, the homologous recombination of the abovedescribed “knock out” and “knock in” constructs is very rare andfrequently such a construct inserts nonhomologously into a random regionof the genome where it has no effect on the gene which has been targetedfor deletion, and where it can potentially recombine so as to disruptanother gene which was otherwise not intended to be altered. Suchnonhomologous recombination events can be selected against by modifyingthe abovementioned knock out and knock in constructs so that they areflanked by negative selectable markers at either end (particularlythrough the use of two allelic variants of the thymidine kinase gene,the polypeptide product of which can be selected against in expressingcell lines in an appropriate tissue culture medium well known in theart—i.e. one containing a drug such as 5-bromodeoxyuridine). Thus apreferred embodiment of such a knock out or knock in construct of theinvention consist of a nucleic acid encoding a negative selectablemarker linked to a nucleic acid encoding a 5′ end of a genomic locuslinked to a nucleic acid of a positive selectable marker which in turnis linked to a nucleic acid encoding a 3′ end of the same genomic locuswhich in turn is linked to a second nucleic acid encoding a negativeselectable marker Nonhomologous recombination between the resultingknock out construct and the genome will usually result in the stableintegration of one or both of these negative selectable marker genes andhence cells which have undergone nonhomologous recombination can beselected against by growth in the appropriate selective media (e.g.media containing a drug such as 5-bromodeoxyuridine for example).Simultaneous selection for the positive selectable marker and againstthe negative selectable marker will result in a vast enrichment forclones in which the knock out construct has recombined homologously atthe locus of the gene intended to be mutated. The presence of thepredicted chromosomal alteration at the targeted gene locus in theresulting knock out stem cell line can be confirmed by means of Southernblot analytical techniques which are well known to those familiar in theart. Alternatively, PCR can be used.

[0164] Each knockout construct to be inserted into the cell must firstbe in the linear form. Therefore, if the knockout construct has beeninserted into a vector (described infra), linearization is accomplishedby digesting the DNA with a suitable restriction endonuclease selectedto cut only within the vector sequence and not within the knockoutconstruct sequence.

[0165] For insertion, the knockout construct is added to the ES cellsunder appropriate conditions for the insertion method chosen, as isknown to the skilled artisan. For example, if the ES cells are to beelectroporated, the ES cells and knockout construct DNA are exposed toan electric pulse using an electroporation machine and following themanufacturer's guidelines for use. After electroporation, the ES cellsare typically allowed to recover under suitable incubation conditions.The cells are then screened for the presence of the knock out constructas explained above. Where more than one construct is to be introducedinto the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

[0166] After suitable ES cells containing the knockout construct in theproper location have been identified by the selection techniquesoutlined above, the cells can be inserted into an embryo. Insertion maybe accomplished in a variety of ways known to the skilled artisan,however a preferred method is by microinjection. For microinjection,about 10-30 cells are collected into a micropipet and injected intoembryos that are at the proper stage of development to permitintegration of the foreign ES cell containing the knockout constructinto the developing embryo. For instance, the transformed ES cells canbe microinjected into blastocytes. The suitable stage of development forthe embryo used for insertion of ES cells is very species dependent,however for mice it is about 3.5 days. The embryos are obtained byperfusing the uterus of pregnant females. Suitable methods foraccomplishing this are known to the skilled artisan, and are set forthby, e.g., Bradley et al. (supra).

[0167] While any embryo of the right stage of development is suitablefor use, preferred embryos are male. In mice, the preferred embryos alsohave genes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo). Thus, for example, if the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

[0168] After the ES cell has been introduced into the embryo, the embryomay be implanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

[0169] Offspring that are born to the foster mother may be screenedinitially for mosaic coat color where the coat color selection strategy(as described above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by Southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross, as well as mice that are known heterozygotes andwild type mice.

[0170] Other means of identifying and characterizing the knockoutoffspring are available. For example, Northern blots can be used toprobe the mRNA for the presence or absence of transcripts encodingeither the gene knocked out, the marker gene, or both. In addition,Western blots can be used to assess the level of expression of the GLC1Agene knocked out in various tissues of the offspring by probing theWestern blot with an antibody against the particular myocilin protein,or an antibody against the marker gene product, where this gene isexpressed. Finally, in situ analysis (such as fixing the cells andlabeling with antibody) and/or FACS (fluorescence activated cellsorting) analysis of various cells from the offspring can be conductedusing suitable antibodies to look for the presence or absence of theknockout construct gene product.

[0171] Yet other methods of making knock-out or disruption transgenicanimals are also generally known. See, for example, Manipulating theMouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g.by homologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a GLC1A-gene can becontrolled by recombinase sequences (described infra).

[0172] Animals containing more than one knockout construct and/or morethan one transgene expression construct are prepared in any of severalways. The preferred manner of preparation is to generate a series ofmammals, each containing one of the desired transgenic phenotypes. Suchanimals are bred together through a series of crosses, backcrosses andselections, to ultimately generate a single animal containing alldesired knockout constructs and/or expression constructs, where theanimal is otherwise congenic (genetically identical) to the wild typeexcept for the presence of the knockout construct(s) and/ortransgene(s).

[0173] A GLC1 A transgene can encode the wild-type form of the protein,or can encode homologs thereof, including both agonists and antagonists,as well as antisense constructs. In preferred embodiments, theexpression of the transgene is restricted to specific subsets of cells,tissues or developmental stages utilizing, for example, cis-actingsequences that control expression in the desired pattern. In the presentinvention, such mosaic expression of a myocilin protein can be essentialfor many forms of lineage analysis and can additionally provide a meansto assess the effects of, for example, lack of GLC1A expression whichmight grossly alter development in small patches of tissue within anotherwise normal embryo. Toward this and, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the transgene in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences.

[0174] Genetic techniques, which allow for the expression of transgenescan be regulated via site-specific genetic manipulation in vivo, areknown to those skilled in the art. For instance, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination of a target sequence. As usedherein, the phrase “target sequence” refers to a nucleotide sequencethat is genetically recombined by a recombinase. The target sequence isflanked by recombinase recognition sequences and is generally eitherexcised or inverted in cells expressing recombinase activity.Recombinase catalyzed recombination events can be designed such thatrecombination of the target sequence results in either the activation orrepression of expression of one of the subject myocilin proteins. Forexample, excision of a target sequence which interferes with theexpression of a recombinant GLC1A gene, such as one which encodes anantagonistic homolog or an antisense transcript, can be designed toactivate expression of that gene. This interference with expression ofthe protein can result from a variety of mechanisms, such as spatialseparation of the GLC1A gene from the promoter element or an internalstop codon. Moreover, the transgene can be made wherein the codingsequence of the gene is flanked by recombinase recognition sequences andis initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

[0175] The transgenic animals of the present invention all includewithin a plurality of their cells a transgene of the present invention,which transgene alters the phenotype of the “host cell” with respect toregulation of cell growth, death and/or differentiation. Since it ispossible to produce transgenic organisms of the invention utilizing oneor more of the transgene constructs described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

[0176] In an illustrative embodiment, either the crelloxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0177] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation expression of a recombinant myocilinprotein can be regulated via control of recombinase expression.

[0178] Use of the cre/loxP recombinase system to regulate expression ofa recombinant myocilin protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant GLC1A gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., a GLC1A gene and recombinase gene.

[0179] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the GLC1A transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

[0180] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding thetrans-activating protein, e.g. a recombinase or a prokaryotic protein,is delivered to the tissue and caused to be expressed, such as in acell-type specific manner. By this method, a GLC1A transgene couldremain silent into adulthood until “turned on” by the introduction ofthe trans-activator.

[0181] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germine ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

[0182] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for micro-injection.In the mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

[0183] Normally, fertilized embryos are incubated in suitable mediauntil the pronuclei appear. At about this time, the nucleotide sequencecomprising the transgene is introduced into the female or malepronucleus as described below. In some species such as mice, the malepronucleus is preferred. It is most preferred that the exogenous geneticmaterial be added to the male DNA complement of the zygote prior to itsbeing processed by the ovum nucleus or the zygote female pronucleus. Itis thought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

[0184] Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

[0185] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0186] For the purposes of this invention a zygote is essentially theformation of a diploid cell which is capable of developing into acomplete organism. Generally, the zygote will be comprised of an eggcontaining a nucleus formed, either naturally or artificially, by thefusion of two haploid nuclei from a gamete or gametes. Thus, the gametenuclei must be ones which are naturally compatible, i.e., ones whichresult in a viable zygote capable of undergoing differentiation anddeveloping into a functioning organism. Generally, a euploid zygote ispreferred. If an aneuploid zygote is obtained, then the number ofchromosomes should not vary by more than one with respect to the euploidnumber of the organism from which either gamete originated.

[0187] In addition to similar biological considerations, physical onesalso govern the amount (e.g., volume) of exogenous genetic materialwhich can be added to the nucleus of the zygote or to the geneticmaterial which forms a part of the zygote nucleus. If no geneticmaterial is removed, then the amount of exogenous genetic material whichcan be added is limited by the amount which will be absorbed withoutbeing physically disruptive. Generally, the volume of exogenous geneticmaterial inserted will not exceed about 10 picoliters. The physicaleffects of addition must not be so great as to physically destroy theviability of the zygote. The biological limit of the number and varietyof DNA sequences will vary depending upon the particular zygote andfunctions of the exogenous genetic material and will be readily apparentto one skilled in the art, because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

[0188] The number of copies of the transgene constructs which are addedto the zygote is dependent upon the total amount of exogenous geneticmaterial added and will be the amount which enables the genetictransformation to occur. Theoretically only one copy is required;however, generally, numerous copies are utilized, for example,1,000-20,000 copies of the transgene construct, in order to insure thatone copy is functional. As regards the present invention, there willoften be an advantage to having more than one functioning copy of eachof the inserted exogenous DNA sequences to enhance the phenotypicexpression of the exogenous DNA sequences.

[0189] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0190] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0191] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0192] Alternative or additional methods for evaluating the presence ofthe transgene include, without limitation, suitable biochemical assayssuch as enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

[0193] Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

[0194] The transgenic animals produced in accordance with the presentinvention will include exogenous genetic material. As set out above, theexogenous genetic material will, in certain embodiments, be a DNAsequence which results in the production of a myocilin protein (eitheragonistic or antagonistic), and antisense transcript, or a myocilinmutant. Further, in such embodiments the sequence will be attached to atranscriptional control element, e.g., a promoter, which preferablyallows the expression of the transgene product in a specific type ofcell.

[0195] Retroviral infection can also be used to introduce transgene intoa non-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0196] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0197] 4.6. Drug Screening Assays for GLC1A Therapeutics

[0198] Based on the discovery of the GLC1A gene and specific mutationsin the gene that correlate with the existence of glaucoma, one of skillin the art is able to use any of a variety of standard assays to screenfor drugs, which will interfere with or otherwise prevent thedevelopment of glaucoma. By addressing the molecular basis of glaucoma,these agents are expected to be superior to existing therapies.

[0199] For example, identification of the precise phenotype associatedwith these mutations can be used to identify functionally importantregions of the protein. These specific mutations can then be used inother experiments which will include overexpression in cell lines andthe creation of transgenic animals. Ideally, one could identifymutations which reproducibly cause glaucoma at very different times inthe person's life and then be able to show that these mutations hadsimilar differences of effect in a cellular expression system or atransgenic animal.

[0200] In addition, proteins that interact with the GLC1A gene productand genes encoding the proteins can now be identified, since proteinsthat interact with GLC1A gene product will be important targets forinvolvement in the pathogenesis of various types of glaucoma.

[0201] Further, studies will be undertaken to discover whether mutationsknown to cause glaucoma in human beings alter protein trafficking intissue culture as well as animal models, since one mechanism throughwhich mutations in the GLC1A gene could cause disease would be to alterthe expression of other important gene products. This can occur byaffecting overall protein trafficking within the cell caused for exampleby increased removal of mutant proteins at the level of the endoplasmicreticulum.

[0202] Further understanding of the pathogenesis of glaucoma is usefulfor identifying new classes of drugs which can be useful in thetreatment of glaucoma. For example, the GLC1A gene has been found to beinduced by exposure of cells to steroids. Therefore, drugs which arecapable of blocking this steroid effect should prove useful forpreventing or delaying the development of glaucoma.

[0203] As further described below, in vitro assays which are suitablefor very high throughput screening of compounds can be performed. As thesimplest example of this approach, one could use antibodies to the GLC1Agene product to develop a simple ELISA assay for the induction of theGLC1A gene product and then perform this assay in a 96 well microtiterplate format to screen a large number of drugs for the efficacy inblocking the steroid induction of the gene product. In this way,automated methods could be used to screen several thousand potentiallytherapeutic compounds for efficacy.

[0204] Also, knowledge of the structure/function of the GLC1A geneimmediately suggests other genes which might be involved in glaucoma.Such clues will come from studies of homology, evolution, evaluation ofstructural motifs within the gene, and genetic studies using analysesdesigned to identify genes causing polygenic disease.

[0205] In the original linkage study described herein, it was recognizedthat 3 of 22 obligate carriers of the glaucoma gene failed to manifest asevere glaucoma phenotype. This information suggests that other genesare capable of mitigating the effect of the GLC1A mutation. One powerfulway to search for such mitigator genes is to express a glaucoma-causinggene in different backgrounds. This can be done by creating transgenicanimals and then breeding the glaucoma-causing gene on different geneticmouse strains. If the phenotype is altered in different strains theseanimals can be back crossed in such a way that the mitigating gene canbe identified.

[0206] Some of the assays mentioned above, will now be described infurther detail below.

[0207] 4.6.1 Cell-Free Assays

[0208] In many drug screening programs which test libraries of compoundsand natural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with proteinswhich may function upstream (including both activators and repressors ofits activity) or to proteins or nucleic acids which may functiondownstream of the myocilin polypeptide, whether they are positively ornegatively regulated by it. To the mixture of the compound and theupstream or downstream element is then added a composition containing amyocilin polypeptide. Detection and quantification of complexes ofmyocilin with it's upstream or downstream elements provide a means fordetermining a compound's efficacy at inhibiting (or potentiating)complex formation between myocilin and a myocilin-binding element. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, isolated and purifiedmyocilin polypeptide is added to a composition containing themyocilin-binding element, and the formation of a complex is quantitatedin the absence of the test compound.

[0209] Complex formation between the myocilin polypeptide and a myocilinbinding element may be detected by a variety of techniques. Modulationof the formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled, fluorescently labeled,or enzymatically labeled myocilin polypeptides, by immunoassay, or bychromatographic detection.

[0210] Typically, it will be desirable to immobilize either myocilin orits binding protein to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of myocilin to an upstreamor downstream element, in the presence and absence of a candidate agent,can be accomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/myocilin (GST/myocilin) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates, e.g. an ³⁵S-labeled, and the testcompound, and the mixture incubated under conditions conducive tocomplex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofmyocilin-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques such as described in theappended examples.

[0211] Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either myocilin orits cognate binding protein can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated myocilin moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withmyocilin but which do not interfere with binding of upstream ordownstream elements can be derivatized to the wells of the plate, andmyocilin trapped in the wells by antibody conjugation. As above,preparations of a myocilin-binding protein and a test compound areincubated in the myocilin-presenting wells of the plate, and the amountof complex trapped in the well can be quantitated. Exemplary methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the myocilin binding element, or which arereactive with myocilin protein and compete with the binding element; aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the binding element, either intrinsic orextrinsic activity. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with themyocilin-BP. To illustrate, the myocilin-BP can be chemicallycross-linked or genetically fused with horseradish peroxidase, and theamount of polypeptide trapped in the complex can be assessed with achromogenic substrate of the enzyme, e.g. 3,3′-diamino-benzadineterahydrochloride or 4-chloro-1-napthol. Likewise, a fusion proteincomprising the polypeptide and glutathione-S-transferase can beprovided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J BiolChem 249:7130).

[0212] For processes which rely on immunodetection for quantitating oneof the proteins trapped in the complex, antibodies against the protein,such as anti-myocilin antibodies, can be used. Alternatively, theprotein to be detected in the complex can be “epitope tagged” in theform of a fusion protein which includes, in addition to the myocilinsequence, a second polypeptide for which antibodies are readilyavailable (e.g. from commercial sources). For instance, the GST fusionproteins described above can also be used for quantification of bindingusing antibodies against the GST moiety. Other useful epitope tagsinclude myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem266:21150-21157) which includes a 10-residue sequence from c-myc, aswell as the pFLAG system (International Biotechnologies, Inc.) or thepEZZ-protein A system (Pharamacia, NJ).

[0213] 4.6.2. Cell Based Assays

[0214] In addition to cell-free assays, such as described above, thereadily available source of mutant and functional GLC1A nucleic acidsand proteins provided by the present invention also facilitates thegeneration of cell-based assays for identifying small moleculeagonists/antagonists and the like. For example, cells can be caused tooverexpress a recombinant myocilin protein in the presence and absenceof a test agent of interest, with the assay scoring for modulation inmyocilin responses by the target cell mediated by the test agent. Aswith the cell-free assays, agents which produce a statisticallysignificant change in myocilin-dependent responses (either inhibition orpotentiation) can be identified. In an illustrative embodiment, theexpression or activity of a myocilin is modulated in cells and theeffects of compounds of interest on the readout of interest (such astissue differentiation, proliferation, tumorigenesis) are measured. Forexample, the expression of genes which are up- or down-regulated inresponse to a myocilin-dependent signal cascade can be assayed. Inpreferred embodiments, the regulatory regions of such genes, e.g., the5′ flanking promoter and enhancer regions, are operably linked to adetectable marker (such as luciferase) which encodes a gene product thatcan be readily detected.

[0215] Exemplary cells or cell lines may be derived from ocular tissue(e.g. trabecular meshwork or ciliary body epithelia); as well as genericmammalian cell lines such as HeLa cells and COS cells, e.g., COS-7(ATCC# CRL-1651). Further, the transgenic animals discussed herein maybe used to generate cell lines containing one or more cell typesinvolved in glaucoma, that can be used as cell culture models for thisdisorder. While primary cultures derived from the glaucomatoustransgenic animals of the invention may be utilized, the generation ofcontinuous cell lines is preferred. For examples of techniques which maybe used to derive a continuous cell line from the transgenic animals,see Small et al., 1985, Mol. Cell Biol. 5:642-648.

[0216] Using these cells, the effect of a test compound on a variety ofend points can be tested including cell proliferation, migration,phagocytosis, adherence and/or biosynthesis (e.g. of extracellularmatrix components). The cells can then be examined for phenotypesassociated with glaucoma, including, but not limited to changes incellular morphology, cell proliferation, cell migration, and celladhesion.

[0217] In the event that the myocilin proteins themselves, or incomplexes with other proteins, are capable of binding DNA and modifyingtranscription of a gene, a transcriptional based assay could be used,for example, in which a myocilin responsive regulatory sequence isoperably linked to a detectable marker gene.

[0218] Monitoring the influence of compounds on cells may be applied notonly in basic drug screening, but also in clinical trials. In suchclinical trials, the expression of a panel of genes may be used as a“read out” of a particular drug's therapeutic effect.

[0219] In yet another aspect of the invention, the subject myocilinpolypeptides can be used to generate a “two hybrid” assay (see, forexample, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), for isolating coding sequences for other cellularproteins which bind to or interact with myocilin (“myocilin-bindingproteins” or “myocilin-bp).

[0220] Briefly, the two hybrid assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for a myocilinpolypeptide. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a sample gene from a cDNA library.If the bait and sample hybrid proteins are able to interact, e.g., forma myocilin-dependent complex, they bring into close proximity the twodomains of the transcriptional activator. This proximity is sufficientto cause transcription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the reporter gene can be detected and usedto score for the interaction of the myocilin and sample proteins.

[0221] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0222] 4.7 Methods of Treating Disease

[0223] In addition to glaucoma, there may be a variety of pathologicalconditions for which myocilin therapeutics of the present invention canbe used in treatment.

[0224] A “myocilin therapeutic,” whether an antagonist or agonist ofwild type myocilin, can be, as appropriate, any of the preparationsdescribed above, including isolated polypeptides, gene therapyconstructs, antisense molecules, peptidomimetics, non-nucleic acid,non-peptidic small molecules, or agents identified in the drug assaysprovided herein.

[0225] As described herein, subjects having certain mutant GLC1A genestend to develop glaucoma. Down-regulation of mutant GLC1A geneexpression and/or a resultant decrease in the activity of a mutantmyocilin protein (e.g. using antisense, ribozyme, triple helix orantibody molecules) and/or up-regulation of a wildtype GLC1 A geneexpression and/or a resultant increase in the activity of a wildtypemyocilin protein (e.g. using gene therapy or protein replacementtherapies) should therefore prove useful in ameliorating diseasesymptoms. Compounds identified as increasing or decreasing GLC1A geneexpression or myocilin protein activity can be administered to a subjectat therapeutically effective dose to treat or ameliorate symptomsassociated with glaucoma.

[0226] 4.7.1. Effective Dose

[0227] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0228] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i e, the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0229] 4.7.2. Formulation and Use

[0230] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

[0231] For such therapy, the oligomers of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection ispreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the oligomers of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theoligomers may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

[0232] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0233] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0234] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0235] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0236] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0237] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0238] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0239] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the oligomersof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art.

[0240] In clinical settings, the gene delivery systems for thetherapeutic GLC1A gene can be introduced into a patient by any of anumber of methods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof Inother embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). A GLC1A gene, such as any one of thesequences represented in the group consisting of SEQ ID NO: 1 or 2, or asequence homologous thereto can be delivered in a gene therapy constructby electroporation using techniques described, for example, by Dev etal. ((1994) Cancer Treat Rev 20:105-115). Gene therapy vectors comprisedof viruses that provide specific effective and highly localizedtreatment of eye diseases are described in Published InternationalPatent Application No. WO 95/34580 to U. Eriksson et al.

[0241] The pharmaceutical preparation of the gene therapy construct canconsist essentially of the gene delivery system in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery system can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

[0242] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0243] 4.8 Predictive Medicine

[0244] The invention further features predictive medicines, which arebased, at least in part, on the identity of the novel GLC1A genes andalterations in the genes and related pathway genes, which affect theexpression level and/or function of the encoded myocilin protein in asubject.

[0245] For example, information obtained using the diagnostic assaysdescribed herein (alone or in conjunction with information on anothergenetic defect, which contributes to the same disease) is useful fordiagnosing or confirming that a symptomatic subject (e.g. a subjectsymptomatic for glaucoma), has a genetic defect (e.g. in a GLC1A gene orin a gene that regulates the expression of an GLC1A gene), which causesor contributes to glaucoma. Alternatively, the information (alone or inconjunction with information on another genetic defect, whichcontributes to the same disease) can be used prognostically forpredicting whether a non-symptomatic subject is likely to developglaucoma. Based on the prognostic information, a doctor can recommend aregimen or therapeutic protocol, useful for preventing or prolongingonset of glaucoma in the individual.

[0246] In addition, knowledge of the particular alteration oralterations resulting in defective or deficient GLC1A genes or proteinsin an individual (the GLC1A genetic profile), alone or in conjunctionwith information on other genetic defects contributing to glaucoma (thegenetic profile of glaucoma) allows customization of therapy to theindividual's genetic profile, the goal of “pharmacogenomics”. Forexample, an individual's GLC1A genetic profile or the genetic profile ofglaucoma, can enable a doctor to: 1) more effectively prescribe a drugthat will address the molecular basis of glaucoma; and 2) betterdetermine the appropriate dosage of a particular drug. For example, theexpression level of myocilin proteins, alone or in conjunction with theexpression level of other genes, known to contribute to glaucoma, can bemeasured in many patients at various stages of the disease to generate atranscriptional or expression profile of glaucoma. Expression patternsof individual patients can then be compared to the expression profile ofglaucoma to determine the appropriate drug and dose to administer to thepatient.

[0247] The ability to target populations expected to show the highestclinical benefit, based on the GLC1A or glaucoma genetic profile, canenable: 1) the repositioning of marketed drugs with disappointing marketresults; 2) the rescue of drug candidates whose clinical development hasbeen discontinued as a result of safety or efficacy limitations, whichare patient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of GLC1A as a marker is useful for optimizing effectivedose).

[0248] These and other methods are described in further detail in thefollowing sections.

[0249] 4.8.1. Prognostic and Diagnostic Assays

[0250] The present methods provide means for determining if a subjecthas (diagnostic) or is at risk of developing (prognostic) glaucoma.

[0251] In one embodiment, the method comprises determining whether asubject has an abnormal GLC1A mRNA and/or myocilin protein level, suchas by Northern blot analysis, reverse transcription-polymerase chainreaction (RT-PCR), in situ hybridization, immunoprecipitation, Westernblot hybridization, or immunohistochemistry. According to the method,cells are obtained from a subject and the level of GLC1A mRNA ormyocilin level is determined and compared to the mRNA or protein levelin a healthy subject. An abnormal level of GLC1A mRNA or myocilintherefor being indicative of an aberrant myocilin bioactivity. Inanother embodiment, the method comprises measuring at least one activityof myocilin. Similarly, the constant of affinity of a myocilin proteinof a subject with a binding partner can be determined. Comparison of theresults obtained with results from similar analysis performed onmyocilin proteins from healthy subjects is indicative of whether asubject has an abnormal myocilin activity.

[0252] In preferred embodiments, the methods for determining whether asubject has or is at risk for developing glaucoma is characterized ascomprising detecting, in a sample of cells from the subject, thepresence or absence of a genetic alteration characterized by at leastone of (i) an alteration affecting the integrity of a gene encoding amyocilin polypeptide, or (ii) the mis-expression of the GLC1A gene. Forexample, such genetic alterations can be detected by ascertaining theexistence of at least one of (i) a deletion of one or more nucleotidesfrom a GLC1A gene, (ii) an addition of one or more nucleotides to aGLC1A gene, (iii) a substitution of one or more nucleotides of a GLC1Agene, (iv) a gross chromosomal rearrangement of a GLC1A gene, (v) agross alteration in the level of a messenger RNA transcript of a GLC1Agene, (vi) aberrant modification of a GLC1A gene, such as of themethylation pattern of the genomic DNA, (vii) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a GLC1A gene,(viii) a non-wild type level of a myocilin polypeptide, (ix) allelicloss of a GLC1A gene, and/or (x) inappropriate post-translationalmodification of a myocilin polypeptide. As set out below, the presentinvention provides a variety of assay techniques for detectingalterations in a GLC1A gene. These methods include, but are not limitedto, methods involving sequence analysis, Southern blot hybridization,restriction enzyme site mapping, and methods involving detection ofabsence of nucleotide pairing between the nucleic acid to be analyzedand a probe. These and other methods are further described infra.

[0253] Specific diseases or disorders, e.g., genetic diseases ordisorders, are associated with specific allelic variants of polymorphicregions of certain genes, which do not necessarily encode a mutatedprotein. Thus, the presence of a specific allelic variant of apolymorphic region of a gene, such as a single nucleotide polymorphism(“SNP”), in a subject can render the subject susceptible to developing aspecific disease or disorder. Polymorphic regions in GLC1A genes, can beidentified by determining the nucleotide sequence of genes inpopulations of individuals. If a polymorphic region, e.g., SNP isidentified, then the link with a specific disease can be determined bystudying specific populations of individuals, e.g., individuals whichdeveloped glaucoma. A polymorphic region can be located in any region ofa gene, e.g., exons, in coding or non-coding regions of exons, introns,and promoter region.

[0254] It is likely that GLC1A genes comprise polymorphic regions,specific alleles of which may be associated with specific diseases orconditions or with an increased likelihood of developing such diseasesor conditions. Thus, the invention provides methods for determining theidentity of the allele or allelic variant of a polymorphic region of aGLC1A gene in a subject, to thereby determine whether the subject has oris at risk of developing a disease or disorder associated with aspecific allelic variant of a polymorphic region.

[0255] In an exemplary embodiment, there is provided a nucleic acidcomposition comprising a nucleic acid probe including a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of a GLC1A gene or naturally occurring mutantsthereof, or 5′ or 3′ flanking sequences or intronic sequences naturallyassociated with the subject GLC1A genes or naturally occurring mutantsthereof The nucleic acid of a cell is rendered accessible forhybridization, the probe is contacted with the nucleic acid of thesample, and the hybridization of the probe to the sample nucleic acid isdetected. Such techniques can be used to detect alterations or allelicvariants at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

[0256] A preferred detection method is allele specific hybridizationusing probes overlapping the mutation or polymorphic site and havingabout 5, 10, 20, 25, or 30 nucleotides around the mutation orpolymorphic region. In a preferred embodiment of the invention, severalprobes capable of hybridizing specifically to allelic variants, such assingle nucleotide polymorphisms, are attached to a solid phase support,e.g., a “chip”. Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. For example a chip can holdup to 250,000 oligonucleotides. Mutation detection analysis using thesechips comprising oligonucleotides, also termed “DNA probe arrays” isdescribed e.g., in Cronin et al. (1996) Human Mutation 7:244. In oneembodiment, a chip comprises all the allelic variants of at least onepolymorphic region of a gene. The solid phase support is then contactedwith a test nucleic acid and hybridization to the specific probes isdetected. Accordingly, the identity of numerous allelic variants of oneor more genes can be identified in a simple hybridization experiment.

[0257] In certain embodiments, detection of the alteration comprisesutilizing the probe/primer in a polymerase chain reaction (PCR) (see,e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACEPCR, or, alternatively, in a ligase chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) PNAS 91:360-364), the latter of which can be particularly usefulfor detecting point mutations in the GLC1A gene (see Abravaya et al.(1995) Nuc Acid Res 23:675-682). In a merely illustrative embodiment,the method includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to a GLC1A gene underconditions such that hybridization and amplification of the GLC1A gene(if present) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

[0258] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0259] In a preferred embodiment of the subject assay, mutations in, orallelic variants, of a GLC1A gene from a sample cell are identified byalterations in restriction enzyme cleavage patterns. For example, sampleand control DNA is isolated, amplified (optionally), digested with oneor more restriction endonucleases, and fragment length sizes aredetermined by gel electrophoresis. Moreover, the use of sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

[0260] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the GLC1Agene and detect mutations by comparing the sequence of the sample GLC1Awith the corresponding wild-type (control) sequence. Exemplarysequencing reactions include those based on techniques developed byMaxim and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger(Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463). It is alsocontemplated that any of a variety of automated sequencing proceduresmay be utilized when performing the subject assays (Biotechniques (1995)19:448), including sequencing by mass spectrometry (see, for example PCTpublication WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162;and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It willbe evident to one skilled in the art that, for certain embodiments, theoccurrence of only one, two or three of the nucleic acid bases need bedetermined in the sequencing reaction. For instance, A-track or thelike, e.g., where only one nucleic acid is detected, can be carried out.

[0261] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetroxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-typeGLC1A sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex as will exist due to basepair mismatches between the control and sample strands. For instance,RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treatedwith S1 nuclease to enzymatically digest the mismatched regions. Inother embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

[0262] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in GLC1A cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aGLC1A sequence, e.g., a wild-type GLC1A sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

[0263] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations or the identity of the allelicvariant of a polymorphic region in GLC1A genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, seealso Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet AnalTech Appl 9:73-79). Single-stranded DNA fragments of sample and controlGLC1A nucleic acids are denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0264] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing agent gradient to identify differences inthe mobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0265] Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labeledtarget DNA.

[0266] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection (Gasparini et al (1992) Mol.Cell Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

[0267] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

[0268] Several techniques based on this OLA method have been developedand can be used to detect specific allelic variants of a polymorphicregion of a GLC1A gene. For example, U.S. Pat. No. 5,593,826 disclosesan OLA using an oligonucleotide having 3′-amino group and a5′-phosphorylated oligonucleotide to form a conjugate having aphosphoramidate linkage. In another variation of OLA described in Tobeet al. ((1996) Nucleic Acids Res 24: 3728), OLA combined with PCRpermits typing of two alleles in a single microtiter well. By markingeach of the allele-specific primers with a unique hapten, i.e.digoxigenin and fluorescein, each OLA reaction can be detected by usinghapten specific antibodies that are labeled with different enzymereporters, alkaline phosphatase or horseradish peroxidase. This systempermits the detection of the two alleles using a high throughput formatthat leads to the production of two different colors.

[0269] The invention further provides methods for detecting singlenucleotide polymorphisms in a GLC1A gene. Because single nucleotidepolymorphisms constitute sites of variation flanked by regions ofinvariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation and it is unnecessary to determine a complete genesequence for each patient. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

[0270] In one embodiment, the single base polymorphism can be detectedby using a specialized exonuclease-resistant nucleotide, as disclosed,e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to themethod, a primer complementary to the allelic sequence immediately 3′ tothe polymorphic site is permitted to hybridize to a target moleculeobtained from a particular animal or human. If the polymorphic site onthe target molecule contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated onto the end of the hybridizedprimer. Such incorporation renders the primer resistant to exonuclease,and thereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method is advantageous, since it does not require the determinationof large amounts of extraneous sequence data.

[0271] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0272] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0273] Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P.et al., Anal. Biochem. 208:171-175 (1993)). These methods differ fromGBA TM in that they all rely on the incorporation of labeleddeoxynucleotides to discriminate between bases at a polymorphic site. Insuch a format, since the signal is proportional to the number ofdeoxynucleotides incorporated, polymorphisms that occur in runs of thesame nucleotide can result in signals that are proportional to thelength of the run (Syvanen, A. -C., et al., Amer. J. Hum. Genet.52:46-59 (1993)).

[0274] For mutations that produce premature termination of proteintranslation, the protein truncation test (PTT) offers an efficientdiagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21;van der Luijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA isinitially isolated from available tissue and reverse-transcribed, andthe segment of interest is amplified by PCR. The products of reversetranscription PCR are then used as a template for nested PCRamplification with a primer that contains an RNA polymerase promoter anda sequence for initiating eukaryotic translation. After amplification ofthe region of interest, the unique motifs incorporated into the primerpermit sequential in vitro transcription and translation of the PCRproducts. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresisof translation products, the appearance of truncated polypeptidessignals the presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

[0275] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid, primer set; and/or antibody reagent described herein,which may be conveniently used, e.g., in clinical settings to diagnosepatients exhibiting symptoms or family history of glaucoma.

[0276] Any cell type or tissue may be utilized in the diagnosticsdescribed below. In a preferred embodiment a bodily fluid, e.g., blood,is obtained from the subject to determine the presence of a mutation orthe identity of the allelic variant of a polymorphic region of a GLC1Agene. A bodily fluid, e.g., blood, can be obtained by known techniques(e.g. venipuncture). Alternatively, nucleic acid tests can be performedon dry samples (e.g. hair or skin). For prenatal diagnosis, fetalnucleic acid samples can be obtained from maternal blood as described inInternational Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

[0277] When using RNA or protein to determine the presence of a mutationor of a specific allelic variant of a polymorphic region of a GLC1Agene, the cells or tissues that may be utilized must express the GLC1Agene. Preferred cells for use in these methods include photoreceptorscells of retina. Alternative cells or tissues that can be used, can beidentified by determining the expression pattern of the specific GLC1Agene in a subject, such as by Northern blot analysis.

[0278] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, NY).

[0279] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0280] Antibodies directed against wild type or mutant myocilinpolypeptides or allelic variants thereof, which are discussed above, mayalso be used in disease diagnostics and prognostics. Such diagnosticmethods, may be used to detect abnormalities in the level of myocilinpolypeptide expression, or abnormalities in the structure and/or tissue,cellular, or subcellular location of a myocilin polypeptide. Structuraldifferences may include, for example, differences in the size,electronegativity, or antigenicity of the mutant myocilin polypeptiderelative to the normal myocilin polypeptide. Protein from the tissue orcell type to be analyzed may easily be detected or isolated usingtechniques which are well known to one of skill in the art, includingbut not limited to western blot analysis. For a detailed explanation ofmethods for carrying out Western blot analysis, see Sambrook et al,1989, supra, at Chapter 18. The protein detection and isolation methodsemployed herein may also be such as those described in Harlow and Lane,for example, (Harlow, E. and Lane, D., 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),which is incorporated herein by reference in its entirety.

[0281] This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody (see below)coupled with light microscopic, flow cytometric, or fluorimetricdetection. The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof myocilin polypeptides. In situ detection may be accomplished byremoving a histological specimen from a patient, and applying thereto alabeled antibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the myocilin polypeptide, but alsoits distribution in the examined tissue. Using the present invention,one of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

[0282] Often a solid phase support or carrier is used as a supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0283] One means for labeling an anti-myocilin polypeptide specificantibody is via linkage to an enzyme and use in an enzyme immunoassay(EIA) (Voller, “The Enzyme Linked Immunosorbent Assay (ELISA)”,Diagnostic Horizons 2:1-7, 1978, Microbiological Associates QuarterlyPublication, Walkersville, Md.; Voller, et al., J. Clin. Pathol.31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio,(ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa,et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzymewhich is bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0284] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type or mutant peptides through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March, 1986, which is incorporated by reference herein). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0285] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescarine.

[0286] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0287] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0288] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0289] Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

[0290] 4.8.2. Pharmacogenomics

[0291] Knowledge of the particular alteration or alterations, resultingin defective or deficient GLC1A genes or proteins in an individual (theGLC1A genetic profile), alone or in conjunction with information onother genetic defects contributing to glaucoma (the genetic profile ofglaucoma) allows a customization of the therapy for glaucoma to theindividual's genetic profile, the goal of “pharmacogenomics”. Forexample, subjects having a specific allele of a GLC1A gene may or maynot exhibit symptoms of glaucoma or be predisposed to developingsymptoms glaucoma. Further, if those subjects are symptomatic, they mayor may not respond to a certain drug, e.g., a specific GLC1Atherapeutic, but may respond to another. Thus, generation of a GLC1Agenetic profile, (e.g., categorization of alterations in GLC1A geneswhich are associated with the development of glaucoma), from apopulation of subjects, who are symptomatic for glaucoma (a glaucomagenetic population profile) and comparison of an individual's GLC1Aprofile to the population profile, permits the selection or design ofdrugs that should be safer and more effective for a particular patientor patient population (i.e., a group of patients having the same geneticalteration).

[0292] For example, a GLC1A population profile can be performed, bydetermining the GLC1A profile, e.g., the identity of GLC1A genes, in apatient population having glaucoma. Optionally, the GLC1A populationprofile can further include information relating to the response of thepopulation to a GLC1A therapeutic, using any of a variety of methods,including, monitoring: 1) the severity of symptoms associated with theGLC1A related disease, 2) GLC1A gene expression level, 3) GLC1A mRNAlevel, and/or 4) GLC1A protein level. and (iii) dividing or categorizingthe population based on the particular genetic alteration or alterationspresent in its GLC1A gene or a GLC1A pathway gene. The GLC1A geneticpopulation profile can also, optionally, indicate those particularalterations in which the patient was either responsive or non-responsiveto a particular therapeutic. This information or population profile, isthen useful for predicting which individuals should respond toparticular drugs, based on their individual GLC1A profile.

[0293] In a preferred embodiment, the GLC1A profile is a transcriptionalor expression level profile and step (i) is comprised of determining theexpression level of GLC1A proteins, alone or in conjunction with theexpression level of other genes, known to contribute to the samedisease. The GLC1A profile can be measured in many patients at variousstages of the disease.

[0294] Pharmacogenomic studies can also be performed using transgenicanimals. For example, one can produce transgenic mice, e.g., asdescribed herein, which contain a specific allelic variant of a GLC1Agene. These mice can be created, e.g., by replacing their wild-typeGLC1A gene with an allele of the human GLC1A gene. The response of thesemice to specific GLC1A therapeutics can then be determined.

[0295] 4.8.3. Monitoring of Effects of GLC1A Therapeutics DuringClinical Trials

[0296] The ability to target populations expected to show the highestclinical benefit, based on the GLC1A or disease genetic profile, canenable: 1) the repositioning of marketed drugs with disappointing marketresults; 2) the rescue of drug candidates whose clinical development hasbeen discontinued as a result of safety or efficacy limitations, whichare patient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of GLC1A as a marker is useful for optimizing effectivedose).

[0297] The treatment of an individual with a GLC1A therapeutic can bemonitored by determining GLC1A characteristics, such as myocilin proteinlevel or activity, GLC1A mRNA level, and/or transcriptional level. Thismeasurements will indicate whether the treatment is effective or whetherit should be adjusted or optimized. Thus, GLC1A can be used as a markerfor the efficacy of a drug during clinical trials.

[0298] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidate, forexample a drug candidate identified by the screening assays describedherein) comprising the steps of (i) obtaining a preadministration samplefrom a subject prior to administration of the agent; (ii) detecting thelevel of expression of a myocilin protein, mRNA, or genomic DNA in thepreadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the myocilin protein, mRNA, or genomic DNAin the post-administration samples; (v) comparing the level ofexpression or activity of the myocilin protein, mRNA, or genomic DNA inthe preadministration sample with the myocilin protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression of a wildtype GLC1A gene or activity of a wildtype myocilinprotein to higher levels than detected. Alternatively, decreasedadministration of the agent may be desirable to decrease expression of amutant GLC1 A gene or activity of a mutant myocilin protein to lowerlevels than detected.

[0299] Cells of a subject may also be obtained before and afteradministration of a GLC1A therapeutic to detect the level of expressionof genes other than GLC1A, to verify that the GLC1A therapeutic does notincrease or decrease the expression of genes which could be deleterious.This can be done, e.g., by using the method of transcriptionalprofiling. Thus, mRNA from cells exposed in vivo to a GLC1A therapeuticand mRNA from the same type of cells that were not exposed to the GLC1Atherapeutic could be reverse transcribed and hybridized to a chipcontaining DNA from numerous genes, to thereby compare the expression ofgenes in cells treated and not treated with a GLC1A therapeutic. If, forexample a GLC1A therapeutic turns on the expression of a proto-oncogenein an individual, use of this particular GLC1A therapeutic may beundesirable.

[0300] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods in Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (1). M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0301] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

[0302] 5.1 Genetic Linkage of Familial Open Angle Glaucoma to Chromosome1q21-q31

[0303] Materials and Methods

[0304] Pedigree

[0305] A family in which five consecutive generations have been affectedwith juvenile-onset, open-angle glaucoma without iridocorneal angleabnormalities was identified. The family comprised descendants of awoman who emigrated from Germany to the midwestern United States in thelate 1800s. The disease state in affected family members included onsetduring the first 3 decades of life, normal anterior chamber angles, highintraocular pressures, lack of systemic or other ocular abnormalities,and need for surgery to control the glaucoma in affected individuals. Atotal of 35 family members at 50% risk for glaucoma had complete eyeexaminations including visual acuity with refraction, slit-lampbiomicroscopy, applanation tomometry, gonioscopy, stereo discphotography and Humphrey, Goldmann or Octopus perimetry. Two otheraffected patients were ascertained by reviewing records of otherophthalmologists. Patients were considered to be affected for linkage ifthey had documented pressures greater than 30 mm Hg and evidence ofoptic nerve or visual field damage; or, if they had intraocularpressures greater than 22 mm Hg and an obviously affected child.Affected family members are characterized by an early age of diagnosis,a normal appearing trabecular meshwork, very high intraocular pressures(often above 50 mm Hg), and relatively pressure-resistant optic nerves.FIG. 1 is a pictorial representation of the pedigree.

[0306] DNA Typing

[0307] Blood samples were obtained from all living affected familymembers as well as six spouses of affected patients with children. 10 mlblood were obtained from each patient in EDTA-containing glass tubes.DNA was prepared from the blood using a non-organic extraction procedure(Grimberg, J. et al. Nucl. Acids Res 17, 8390 (1989)). Short tandemrepeat polymorphisms (STRPs) distributed across the entire autosomalgenome were selected from the literature or from those kindly providedby J. L. Weber. The majority were [dC-dA]-[dG-dT] dinucleotide repeats.Oligonucleotide primers flanking each STRP were synthesized usingstandard phosphoramidite chemistry (Applied Biosystems model 391 DNAsynthesizer). Amplification of each STRP was performed with 50 ng. ofeach patient's DNA in a 8.35 l PCR containing each of the following:1.25 l 10× buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl₂,0.01% w/v gelatin), 300 M each of dCTP, dGTP and dTTP, 37M dATP, 50pmoles each primer, 0.25 l-³⁵S-dATP (Amersham, >1000 Ci mmol⁻¹), and0.25 U Taq polymerase (Perkin-Elmer/Cetus). Samples were incubated in aDNA thermocycler (Perkin-Elmer/Cetus) for 35 cycles under the followingconditions: 94C for 30 s, 55C for 30 s, and 72C for 30 s. Followingamplification, 51 of stop solution (95% formamide, 10 mM NaOH, 0.05%Bromophenol Blue, 0.05% Xylene Cyanol) was added to each sample.Following denaturation for 3 min at 95C, 5 l of each sample wasimmediately loaded onto prewarmed polyacrylamide gels (6%polyacrylamide, 7 M urea) and electrophoresed for 3-4h. Gels were thenplaced on Whatman, 3 mm paper and dried in a slab gel dryer.Autoradiographs were created by exposing Kodak Xomat AR film to thedried gels for 24-36h.

[0308] Linkage Analysis

[0309] Genotypic data from the autoradiographs were entered into aMacintosh computer. A Hypercard-based program (Nichols, B E et al., Am JHum Genet 51 A369 (1992)) was used to store and retrieve marker data aswell as to export it to a DOS-compatible machine for analysis with thecomputer program LINKAGE (version 5.1) (Lathrop, G M and LaLouel, J M359, 794-801 (1992)). Allele frequencies were assumed to be equal foreach marker. The MLINK routine was used for pairwise analysis. Therelative odds of all possible orders of the disease and two markers(D1S191 and D1S194) was performed under the ILINK program. Significanceof linkage was evaluated using the standard criterion (Z_(max)>3.0).

[0310] Results

[0311] Clinical Findings

[0312] All of the 37 family members studied were at 50% risk of havingthe disease because of a known affected parent or sibling. Nineteen ofthese patients had elevated intraocular pressures and visual fielddefects consistent with the diagnosis of primary open angle glaucoma.Three more patients had moderately elevated intraocular pressures andobviously affected children.

[0313] Linkage Analysis

[0314] Over 90 short tandem repeat polymorphisms were typed the familybefore linkage was detected with markers that map to the long arm ofchromosome 1. Two-point maximum likelihood calculations using allavailable family members and 33 chromosome 1 markers revealedsignificant linkage to eight of them (Table 2). D1S212 was fullyinformative for all affected members of the family, and pairwise linkageanalysis produced a lod score of 6.5 (=0), Multipoint linkage analysisdid not add to the peak lod score. The glaucoma locus was thereforedetermined to be located in a region of about 20 centiMorgans (cM) insize between D1S191 and D1S194. Both of these markers demonstratedmultiple recombinants (two and three, respectively) in affectedindividuals in the family. The order D1S191-glaucoma-D1S194 was morethan 1,000 times more likely than the other two possible orders. TABLE 6Pairwise linkage data Recombination Fraction 0.15 0.20 0.25 0.30 0.40Z_(max) Locus {circumflex over (θ)} D1S212 6.0 5.4 4.8 4.2 3.6 2.9 1.46.5 0.00 1 D1S215 5.1 4.6 4.0 3.5 2.9 2.3 1.0 5.6 0.00 0 D1S218 4.7 4.33.8 3.3 2.7 2.2 1.0 5.2 0.00 1 D1S238 4.4 4.2 3.9 3.4 2.9 2.4 1.2 4.40.04 1 D1S117 3.8 3.6 3.3 2.8 2.3 1.8 0.7 3.8 0.04 1q D1S104 3.2 2.9 2.62.3 2.0 1.6 0.7 3.4 0.00 1q21-q23 D1S191 3.0 3.2 3.0 2.7 2.4 1.9 0.9 3.20.09 1 D1S196 2.9 2.6 2.3 2.0 1.6 1.3 0.5 3.1 0.00 1

[0315] Table 6 Pairwise Linkage Data

[0316] Recombination Fraction

[0317] 0.05 0.19

[0318] 5.2 Genetic Fine Mapping of the Juvenile Primary Open AngleGlaucoma Locus and Identification and Characterization of a GlaucomaGene

[0319] Once primary linkage has been identified, the next step inidentifying any disease gene by positional cloning is the narrowing ofthe candidate locus to the smallest possible genetic region. The initialstudy described in Example 5.1 demonstrated that a primary open angleglaucoma gene lies within an approximately 20 cM region flanked bymarkers D1S194 and D1S191 on chromosome 1q. Additional markers andfamilies were obtained and used to refine the genetic locus to a 2.5 cMregion using two of these families. The third family should allow theinterval to be further narrowed.

[0320] In addition to the family resources, polymorphic DNA markers andgenetic maps were used to refine the 1q glaucoma locus. Using STRPs, thegenotype of each family member was determined. Amplification of eachSTRP was performed using the following protocol:

[0321] 1) Dilute genomic DNA (about 1 g/l) 1/50 i.e. 201 “stock” DNA and980 dd H₂O.

[0322] 2) Use 2.5 l of “dilute” DNA as template for PCR

[0323] 3) Prepare PCR reaction mix as follows:

[0324] 1.25 l 10× Buffer (Stratagene)

[0325] 0.12l of each primer (50 pmoles each primer)

[0326] 0.5 l dNTPs (5 mM C, T, & G and 0.625 mM A “cold”)

[0327] 3.5 l dd H₂O

[0328]0.25 l ³⁵S-dATP

[0329] 0.1 l Taq polymerase oil (one drop)

[0330] 4) Perform PCR at optimal conditions for given primers (usually94 30 s, 55 30s and 72 30 s) and run for 35 cycles.

[0331] 5) Add 5 l stop solution (95% formamide, 10 mM NaOH, 0.05%bromophenol blue, 0.05% xylene cyanol) to each tube.

[0332] 6) Denature samples at 95C for 3 minutes and load immediatelyonto a prewarmed polyacrylamide gel.

[0333] 7) Dry gels on Whatmann paper and expose autoradiography film for1-2 days.

[0334] Where possible, multiple loadings of different STRPs on gels wereperformed. Up to 6 markers per gel have been successfully loaded. Inaddition, the PCR amplification (up to three markers) have beensuccessfully multiplexed. The juvenile glaucoma gene is believed to liebetween markers AFM238 and AT3 (an 8 centimorgan interval) based onobserved recombinations within the families studied. Haplotypic analysisbetween families has further narrowed this interval to the 2 centimorganinterval between D1S210 and AT3.

[0335] Since the genetic interval has been narrowed significantlyphysical mapping strategies can be used. The closest flanking markers toscreen total human genomic yeast artificial chromosome (YAC) librariesto identify YACs mapping to the region of interest. The CEPH and CEPHmega-YAC libraries can be used for this purpose (available from theCentre d'Etude du Polymorphisme Humain (CEPH) Paris, France). Forty-fourpercent of the clones in the CEPH mega-YAC library have an average sizeof 560 kb, an additional 21% have an average size of 800 kb, and 35%have an average size of 120 kb. This library is available in a griddedmicro-titer plate format such that only 50-200 PCR reactions need to beperformed using a specific sequence tagged site (STS) to identify aunique YAC containing the STS. The YAC contigs identified by CEPH havebeen used to begin constructing a contig across the 1q candidate region(see FIG. 3). YAC contigs using YAC ends can be constructed to identifyadditional YACs. YAC ends can be rescued using anchored PCR (Riley, J.et al (1990) Nucleic Acids Res 18:2887-2890), the ends can then besequenced and the sequence can be used to develop a sequence tagged site(STS). The STS can be used to rescreen the YAC library to obtain anoverlapping adjacent YAC.

[0336] Because some YACs have been shown to be chimeric or to containdeletions or rearrangements, particularly those from the mega YAClibrary, the correctness of each YAC contig should be verified byconstructing a pulse field map of the region. In addition, chimeric YACsare minimized by ensuring that the YAC maps to a single chromosome byfluorescent in situ hybridization (FISH) or that the two YAC ends map tothe same chromosome using monochromosomal somatic cell hybrids (NIGMsPanel 2). In addition, the YAC chimera problem can be minimized by notrelying on any single YAC to span a given chromosome segment, but ratherby obtaining at least two overlapping independent YACs to ensurecoverage of a given region.

[0337] Once a YAC contig spanning the candidate region has beenisolated, this reagent can be used to generate additional geneticmarkers for potentially finer genetic mapping. In addition, the YACs canbe used to make higher resolution physical mapping reagents such asregion specific lambda and cosmid clones. Lambda and cosmid clones canbe used for isolation of candidate genes. A modification of “exontrapping” (Duyk, G. M. (1990) Proc Natl Acad Sci USA 87:8995-8999) knownas exon amplification (Buckler, A. J. (1991) Proc Natl Acad Sci USA88:4005-4009) can be used to identify exons from genes within theregion. Exons trapped from the candidate region can be used as probes toscreen eye cDNA libraries to isolate cDNAs. Where necessary, otherstrategies can be utilized to identify genes in genomic DNA includingscreening cDNA libraries with YAC fragments subcloned into cosmids, zooblot analysis, coincidence cloning strategies such as direct selectionof cDNAs with biotin-streptavidin tagged cosmid clones (Morgan, J. G. etal (1992) Nucleic Acid Res 20 (19):5173-5179), and HTF island analysis(Bird, A. P. (1987) Trends Genet 3:342-247). Promising genes will befurther evaluated by searching for mutations using GC-clamped denaturinggradient gel electrophoresis (Sheffield, V. C. et al (1989) Genomics16:325-332), single strand conformational gel polymorphism (SSCP)analysis (Orita, M. et al (1989) Proc Natl Acad Sci USA 86:2766-2770)and direct DNA sequencing.

[0338] 5.3 Primer Pairs for Use in Identifying Subjects Having aPredisposition to Glaucoma

[0339] Two primer pairs that can be used in conjunction with thepolymerase chain reaction to amplify a 190 base pair sequence from humangenomic DNA that harbors mutations causing glaucoma (primers 1 and 2 inTable 7) have been identified. TABLE 7 Primer 1forward-ATACTGCCTAGGCCACTGGA (SEQ ID NO.12) reverse-CAATGTCCGTGTAGCCACC(SEQ ID NO.13) Primer 2 forward-GAACTCGAACAAACCTGGGA (SEQ ID NO.14)reverse-CATGCTGCTGTACTTATAGCGG (SEQ ID NO.15)

[0340] These primers were used to screen 410 patients with glaucoma and81 normal individuals. Four amino acid altering sequence changes weredetected in a total of 12 glaucoma patients (2.9%). No amino acidaltering sequence changes were observed in the normal individuals.

[0341] The prevalence of mutations in the segment of DNA amplified bythese primer pairs suggest that use of these primers in conjunction withan appropriate detection method can be used to identify a predispositionto glaucoma in approximately 100 thousand patients in the United Statesalone.

[0342] 5.4 Additional Primer Pairs and Their Use in Identifying SubjectsHaving a Predisposition to Glaucoma

[0343] The study was approved by the Human Subjects Review Committee atthe University of Iowa and informed consent was obtained from all studyparticipants. Primary open angle glaucoma was defined as the presence ofan intraocular pressure over 21 mm Hg as well as evidence ofglaucomatous optic nerve head damage. Visible optic nerve head damagealone was accepted if there was documented enlargement of the opticnerve head cup. Otherwise, both a large optic nerve head cup with a thinneural rim and characteristic optic nerve related visual field loss wererequired. Patients were excluded if they had a history of eye surgeryprior to the diagnosis of glaucoma or evidence of secondary glaucoma,such as exfoliation or pigment dispersion. Normal volunteers were over40 years of age, had intraocular pressures under 20 mm Hg, and had nofamily or personal history of glaucoma. 716 unrelated patients affectedwith primary open angle glaucoma (POAG) and 91 volunteers were screenedfor mutations in the coding sequence of the GLC1A gene. This wasaccomplished with an electrophoretic procedure known as single strandconformation polymorphism analysis (SSCP). The sequences of theoligonucleotide primers used for the GLC1A assay are presented in Table8. TABLE 8 Primer Pairs Exon Forward Primer Reverse Primer 1 SEQ ID No.16 SEQ ID No. 17 1 SEQ ID No. 18 SEQ ID No. 19 1 SEQ ID No. 20 SEQ IDNo. 21 1 SEQ ID No. 22 SEQ ID No. 23 1 SEQ ID No. 24 SEQ ID No. 25 1 SEQID No. 26 SEQ ID No. 27 2 SEQ ID No. 28 SEQ ID No. 29 3 SEQ ID No. 30SEQ ID No. 31 3 SEQ ID No. 32 SEQ ID No. 33 3 SEQ ID No. 34 SEQ ID No.35 3 SEQ ID No. 36 SEQ ID No. 37 3 SEQ ID No. 38 SEQ ID No. 39 3 SEQ IDNo. 40 SEQ ID No. 41

[0344] Mutations were confirmed with automated DNA sequencing. 227 ofthe patients (32%) were ascertained because of a positive family historyof glaucoma while 402 (56%) were ascertained consecutively in a singleglaucoma clinic (the University of Iowa). Overall, 563 of the patientswere ascertained in Iowa, 97 in Australia and the remainder fromelsewhere in the United States. All of the normal volunteers werecollected in Iowa. More than 75% of the patients in each group wereCaucasian. A portion of the GLC1A gene had been previously evaluated formutations in 330 of these same glaucoma patients and all 91 normalvolunteers (see above). However, in this study, the entire coding regionwas evaluated. An additional 505 unrelated control individuals with anunknown glaucoma status were also evaluated for sequence changes. Threehundred and eighty of these control patients had been previouslyscreened for mutations in a portion of exon 3. 184 of these generalpopulation controls were connected in Iowa and 13 in Australia. Familymembers of the probands found to harbor GLC1A sequence changes were alsoevaluated for mutations. Efforts were made to examine or review themedical records of all molecularly affected family members. The age ofonset and the highest recorded intraocular pressures were associatedwith six different mutations were evaluated with a Kruskal-Wallisnon-parametric analysis of variance. All p values were two-tailed. Inthe four largest families, co-segregation of a GLC1A mutation and thedisease phenotype was evaluated with the LOD score method as describedabove

[0345] 5.5 Cloning and Sequencing Human and Mouse GLC1A and NorthernBlot Analysis of Expression

[0346] BAC screening. BAC clones containing the human GLC1A gene wereidentified by screening human BAC library pools (Research Genetics,Huntsville, Ala.) with a PCR-based assay. One microliter of BAC pool DNAwas used as template in an 8.35 μl PCR reaction containing 1.25 μl of10× buffer (100 mM tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl₂);deoxynucleotides dCTP, dATP, dTTP, and dGTP (300 μM each); 1 pmol ofeach primer; and 0.25 units of Taq polymerase (Boehringer Mannheim,Indianapolis, Ind.). The primers used in the screening assay werespecific for exon three of GLC1A (FWD: 5′ ATACTGCCTAGGCCACTGGA 3′ (SEQID No. 34) and REV: 5 CAATGTCCGTGTAGCCACC 3′ (SEQ ID No. 35)). Sampleswere denatured at 94° C. for 5 minutes and incubated for 35 cycles at94° C. 30 s, 55° C. 30 s, 72° C. 30 s in a DNA thermocycler (Omnigene,Teddington, Middlesex, UK). After amplification, 5 μl of stop solution(95% formamide, 10 mM NaOH 0.5% bromophenyl blue, 0.05% xylene cyanol)were added. Amplification products were electrophoresed on 6%polyacrylamide-5% glycerol gels at 50 W for approximately 2 hours. Afterelectrophoresis, gels were stained with silver nitrate (13assam 1991). ABAC containing the mouse GLC1A orthologue was identified by screeningthe mouse 129 BAC library pools Research Genetics, Huntsville Ala.).Primers specific for exon three of the human GLC1A gene (FWD: 5′TGGCTACCACGGACAGTTC 3′ (SEQ ID No. 36) and REV: 5′ CATTGGCCACTGACTGCTTA3′ (SEQ ID No. 37) were used for a primary PCR-based screen as describedabove. The primary screen identified sub-pools of BACs which containedthe mouse GLC1A gene. Filters blotted with the BACs in the subpools(Research Genetics, Huntsville, Ala.) were screened by hybridizationwith a digoxigenin probe using the Genius System hybridization kit(Boehringer Mannheim, Indianapolis, Ind.). Digoxigenin labeled probe forhybridization was generated by PCR amplifying 50 ng of mouse 129 DNA ina 25 μl reaction containing 3.75 μl of 10× buffer; 1.5 μl of labelingdNTP mixture (1 mM dATP, 1 mM dCTP, 1 mM dGTP 0.65 mM dTTP, and 0.35 mMof digoxigenin conjugated dUTP); 7.6 pmoles each of FWD and REV primer;and 1.25 units of Taq polymerase (Boehringer Mannheim, Indianapolis,Ind.). PCR reaction conditions were as described above. Hybridizationconditions were as recommended by the manufacturer.

[0347] The human GLC1A cDNA sequence was used to select PCR primers thatproduced an amplification product of identical size when using bothhuman and mouse genomic DNA as template. The amplification products weresequenced to confirm that they were from the human GLC1A gene and themouse orthologue of this gene. The PCR primers were then used to screenboth a human and mouse BAC library. Both human and mouse BACs containingthe GLC1A gene were identified, subcloned into plasmids, and severalclones covering each GLC1A gene were identified. These subclones wereused to generate both human and mouse genomic GLC1A sequence.

[0348] Subcloning.

[0349] The mouse and human BACs containing the GLC1A gene were digestedwith either EcoR1, Aval, Acc1, or BamH1 and ligated into either pT7-blue(Novagen, Miwaukee, Wis.) or pUC19.

[0350] Sequencing.

[0351] PCR products and BAC subclones were sequenced with fluorescentdideoxynucleotides on an Applied Biosystems (ABI) model 373 or 377automated sequencer.

[0352] GLC1A CA Repeat Polymorphisms.

[0353] The CA repeat polymorphism upstream of the GLC1A gene was PCRamplified with primers 5′-TTCCTTCAGGTTGGGAGATG-3′ (SEQ ID No. 42) and5′-GAGAGCACCAGGAGATGGAG-3′ (SEQ ID No. 43). The PCR reaction conditionswere as described in the BAC screening section. Allele frequencies forthe upstream polymorphism are: Allele 1, 1.1%; Allele 2, 2.2%; Allele 3,48.9%; Allele 4, 1.1%; Allele 5, 21.1%; Allele 6, 25.6%. Allelefrequencies for the downstream polymorphism are: Allele 1, 25.3%; Allele2, 13%, Allele 3, 60.3%, Allele 4, 1 .4%.

[0354] Sequence Comparison.

[0355] DNA sequences were aligned and contigs were formed using theSequencher DNA analysis package (DNA Codes, Ann Arbor, Mich.). Putativeenhancer and promoter elements were identified using the internetresource TESS (http://agave.humgen.upenn.edu/utess/) and thetranscription factor binding site data set TRANSFAC v3.2. The predictedprotein sequence was analyzed with PROSITE, Tmpred, NetOgly, and SignalPsoftware packages available on the internet athttp://expasy.hcuge.chsprot/prosite.html;http://ulrec3.unil.ch/software/TMPED_form.html;http://genome.cbs.dtu.dk/services/netOGLYC/;http://www.cbs.dtu.dk/services/SignalP/. Data base searches forexpression of the GLC1A gene used the program BLAST and the data basesdbest and NR available on the internet athttp://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-blast?Jform=0.

[0356] Northern Blot Analysis.

[0357] Human Multiple Tissue Northern (MTN) blots (Clontech, SanFrancisco, Calif.) were probed either with the entire human GLC1A cDNAsequence or with a section of exon three of the human GLC1A genecorresponding to codon 315 to the termination site. The probes werelabeled with ³²P-(dCTP) using Ready-To-Go DNA Labeling Beads (-dCTP)(Pharmacia Biotech, Piscataway, N.J.). Hybridization was for 16 hours at42° C. in 50% formamide, 5× standard saline citrate (5× SSC: 0.75Msodium chloride, 0.075M sodium acetate), 1× Denhardt's solution, 20 mMphosphate buffer (pH 7.5), 1% sodium dodecyl sulfate (SDS), 100 μg/mlsalmon sperm DNA, and 10% dextran sulfate. Following hybridization,blots were washed twice at room temperature in 1× SSC, rinsed twice in1X SSC/1% SDS at 65° C., and washed once in 0.1× SSC, 0.1% SDS toconfirm the specificity of the hybridization. Autoradiography wasperformed with Kodak XAR-5 film at −70° C. with DuPont Cronex LightningPlus intensifying screens (DuPont, Wilmington, Del.).

1 43 1 2800 DNA Artificial Sequence Description of Artificial Sequence;Note = Synthetic construct 1 agcgcagggg aggagaagaa aagagaggga tagtgtatgagcaagaaaga cagattcatt 60 caagggcagt gggaattgac cacagggatt atagtccacgtgatcctggg ttctaggagg 120 cagggctata ttgtgggggg aaaaaatcag ttcaagggaagtcgggagac ctgatttcta 180 atactatatt tttcctttac aagctgagta attctgagcaagtcacaagg tagtaactga 240 ggctgtaaga ttacttagtt tctccttatt aggaactctttttctctgtg gagttagcag 300 cacaagggca atcccgtttc ttttaacagg aagaaaacattcctaagagt aaagccaaac 360 agattcaagc ctaggtcttg ctgactatat gattggttttttgaaaaatc atttcagcga 420 tgtttactat ctgattcaga aaatgagact agtaccctttggtcagctgt aaacaaacac 480 ccatttgtaa atgtctcaag ttcaggctta actgcagaaccaatcaaata agaatagaat 540 ctttagagca aactgtgttt ctccactctg gaggtgagtctgccagggca gtttggaaat 600 atttacttca caagtattga cactgttgtt ggtattaacaacataaagtt gctcaaaggc 660 aatcattatt tcaagtggct taaagttact tctgacagttttggtatatt tattggctat 720 tgccatttgc tttttgtttt ttctctttgg gtttattaatgtaaagcagg gattattaac 780 ctacagtcca gaaagcctgt gaatttgaat gaggaaaaaattacattttt gtttttacca 840 ccttctaact aaatttaaca ttttattcca ttgcgaatagagccataaac tcaaagtggt 900 aataacagta cctgtgattt tgtcattacc aatagaaatcacagacattt tatactatat 960 tacagttgtt gcagatacgt tgtaagtgaa atatttatactcaaaactac tttgaaatta 1020 gacctcctgc tggatcttgt ttttaacata ttaataaaacatgtttaaaa ttttgatatt 1080 ttgataatca tatttcatta tcatttgttt cctttgtaatctatatttta tatatttgaa 1140 aacatctttc tgagaagagt tccccagatt tcaccaatgaggttcttggc atgcacacac 1200 acagagtaag aactgattta gaggctaaca ttgacattggtgcctgagat gcaagactga 1260 aattagaaag ttctcccaaa gatacacagt tgttttaaagctaggggtga ggggggaaat 1320 ctgccgcttc tataggaatg ctctccctgg agcctggtagggtgctgtcc ttgtgttctg 1380 gctggctgtt atttttctct gtccctgcta cgtcttaaaggacttgtttg gatctccagt 1440 tcctagcata gtgcctggca cagtgcaggt tctcaatgagtttgcagagt gaatggaaat 1500 ataaactaga aatatatcct tgttgaaatc agcacaccagtagtcctggt gtaagtgtgt 1560 gtacgtgtgt gtgtgtgtgt gtgtgtgtgt gtaaaaccaggtggagatat aggaactatt 1620 attggggtat gggtgcataa attgggatgt tctttttaaaaagaaactcc aaacagactt 1680 ctggaaggtt attttctaag aatcttgctg gcagcgtgaaggcaaccccc ctgtgcacag 1740 ccccacccag cctcacgtgg ccacctctgt cttcccccatgaagggctgg ctccccagta 1800 tatataaacc tctctggagc tcgggcatga gccagcaaggccacccatcc aggcacctct 1860 cagcacagca gagctttcca gaggaagcct caccaagcctctgcaatgag gttcttctgt 1920 gcacgttgct gcagctttgg gcctgagatg ccagctgtccagctgctgct tctggcctgc 1980 ctggtgtggg atgtgggggc caggacagct cagctcaggaaggccaatga ccagagtggc 2040 cgatgccagt ataccttcag tgtggccagt cccaatgaatccagctgccc agagcagagc 2100 caggccatgt cagtcatcca taacttacag agagacagcagcacccaacg cttagacctg 2160 gaggccacca aagctcgact cagctccctg gagagcctcctccaccaatt gaccttggac 2220 caggctgcca ggccccagga gacccaggag gggctgcagagggagctggg caccctgagg 2280 cgggagcggg accagctgga aacccaaacc agagagttggagactgccta cagcaacctc 2340 ctccgagaca agtcagttct ggaggaagag aagaagcgactaaggcaaga aaatgagaat 2400 ctggccagga ggttggaaag cagcagccag gaggtagcaaggctgagaag gggccagtgt 2460 ccccagaccc gagacactgc tcgggctgtg ccaccaggctccagagaagg taagaatgca 2520 gagtgggggg actctgagtt cagcaggtga tatggctcgtagtgacctgc tacaggcgct 2580 ccaggcctcc ctgcctgccc tttctcctag agactgcacagctagcacaa gacagatgaa 2640 ttaaggaaag cacagcgatc accttcaagt attactagtaatttagctcc tgagagcttc 2700 atttagatta gtggttcaga gttcttgtgc ccctccatgtcagttttcac agtccatagc 2760 aaaaggagaa ataaaaggac cgggtgagat gtgtctgcat2800 2 680 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 2 caccatgttg gccaggctgg tctcgaactc ctgacctcaggtgatccgcc tgcctcggcc 60 tcccaaagtg ctgggattac aggcatgagc caccacgcctggccggcagc ctatttaaat 120 gtcatcctca acatagtcaa tccttgggcc attttttcttacagtaaaat tttgtctctt 180 tcttttaatg cagtttctac gtggaatttg gacactttggccttccagga actgaagtcc 240 gagctaactg aagttcctgc ttcccgaatt ttgaaggagagcccatctgg ctatctcagg 300 agtggagagg gagacaccgg tatgaagtta agtttcttcccttttgtgcc cacatggtct 360 ttattcatgt ctagtgctgt gttcagagaa tcagtatagggtaaatgccc acccaagggg 420 gaaattaact tccctgggag cagagggagg ggaggagaagaggaacagaa ctctctctct 480 ctctctgttc ccttgtcaga gcaggtctgc aggagtcagcctttccctaa caaagccctc 540 tatcctatca cccacacttg ggaggctggg ctgggctgcacagggcaaga tgagagatgt 600 gttgatttca tccacttgat tgtcatgtag aattagatatacttgagaag ttacattttt 660 cagtagcgcc ttcatatctt 680 3 2000 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 3 cttacaactg atactgagtg aattgtactt taaatatttt atagctcccactcccatgca 60 tgcccctcag tgatagcaat aattgtcaat aacatgaaac acagattgatcatatagcat 120 ttaccatata tttactctat accaagcact taacatatat aattacatttaaaatttaca 180 acagccctac tacccaaaac actattagta tcccctttta cacatgcgataactgaggcg 240 tagagagcta agtaacttac tgaaagtcac acagccagcg ggtggtagagcctagcttta 300 aacccagacg atttgtctcc agggctgtca catctactgg ctctgccaagcttccgcatg 360 atcattgtct gtgtttggaa agattatgga ttaagtggtg cttcgttttcttttctgaat 420 ttaccaggat gtggagaact agtttgggta ggagagcctc tcacgctgagaacagcagaa 480 acaattactg gcaagtatgg tgtgtggatg cgagacccca agcccacctacccctacacc 540 caggagacca cgtggagaat cgacacagtt ggcacggatg tccgccaggtttttgagtat 600 gacctcatca gccagtttat gcagggctac ccttctaagg ttcacatactgcctaggcca 660 ctggaaagca cgggtgctgt ggtgtactcg gggagcctct atttccagggcgctgagtcc 720 agaactgtca taagatatga gctgaatacc gagacagtga aggctgagaaggaaatccct 780 ggagctggct accacggaca gttcccgtat tcttggggtg gctacacggacattgacttg 840 gctgtggatg aagcaggcct ctgggtcatt tacagcaccg atgaggccaaaggtgccatt 900 gtcctctcca aactgaaccc agagaatctg gaactcgaac aaacctgggagacaaacatc 960 cgtaagcagt cagtcgccaa tgccttcatc atctgtggca ccttgtacaccgtcagcagc 1020 tacacctcag cagatgctac cgtcaacttt gcttatgaca caggcacaggtatcagcaag 1080 accctgacca tcccattcaa gaaccgctat aagtacagca gcatgattgactacaacccc 1140 ctggagaaga agctctttgc ctgggacaac ttgaacatgg tcacttatgacatcaagctc 1200 tccaagatgt gaaaagcctc caagctgtac aggcaatggc agaaggagatgctcagggct 1260 cctgggggga gcaggctgaa gggagagcca gccagccagg gcccaggcagctttgactgc 1320 tttccaagtt ttcattaatc cagaaggatg aacatggtca ccatctaactattcaggaat 1380 tgtagtctga gggcgtagac aatttcatat aataaatatc ctttatcttctgtcagcatt 1440 tatgggatgt ttaatgacat agttcaagtt ttcttgtgat ttggggcaaaagctgtaagg 1500 cataatagtt tcttcctgaa aaccattgct cttgcatgtt acatggttaccacaagccac 1560 aataaaaagc ataacttcta aaggaagcag aatagctcct ctggccagcatcgaatataa 1620 gtaagatgca tttactacag ttggcttcta atgcttcaga tagaatacagttgggtctca 1680 cataaccctt tacattgtga aataaaattt tcttacccaa cgttctcttccttgaacttt 1740 gtgggaatct ttgcttaaga gaaggatata gattccaacc atcaggtaattccttcaggt 1800 tgggagatgt gattgcagga tgttaaaggt ggtgtgtgtg tgtgtgtgtgtgtgtgtaac 1860 tgagaggctt gtgcctggtt ttgaggtgct gcccaggatg acgccaagcaaatagcagca 1920 tccacacttt cccacctcca tctcctggtg ctctcggcac taccggagcaatctttccat 1980 ctctcccctg aacccaccct 2000 4 2800 DNA ArtificialSequence Description of Artificial Sequence; Note = synthetic construct4 tacctggtac ttgttggctg gccaatctaa ccaaatcagt gatccccaag ctcagcgaga 60caatccgtct caaaaaaaca aagtggagaa tgaaagaaga caacgcctga cataagcctc 120tagctcacac acacacacac acacacacgc ctatacacat gagtgtgcac ccacccaggt 180gaacgcagat gcacacatac cccacccaca caagaatgga tttagagcaa gaggcacttg 240ctcagtcttc aggcgaatct gctatgggaa catcagagaa atttatcaca cagatatcac 300aaatgctatt attagtatct gagaaccaag ttgctcaaat gcaaatgttg ctctaaggaa 360cccatgaggg ggcagtgagg tggctgagag ggggaggtgc ttagtgagca ggccttacag 420actgaggtca gtccctaaag cccatgccag gaggagagaa ctggacccca aaagttgtcc 480tctgaccaca acacggcatg catggcccat gtgtgctcat atacccccca tatgagcaca 540caccagtaag taaacattta taaagatgtt catgaggctt ccacgcacac actggcttat 600gtgaacttct gacaagcctt ggtacttggt acttggttct cctgcttggt tttggttttt 660ttcatttatc ttattttttt atttggagga aggtgtgtgt gtgtgtgtgt ctctctgtgt 720gtgtgtctgt gtgtgtgtgt gtgtgttgtt gttgttgttg ttgttgacag tttctttttt 780taggagaagt ctcattatac tgcccagttg ttcttgaact ctttttgaga cttaacaatt 840cccttacatt gcattcaaag tagtgggctc tctttgaaaa gggagtacta ttagcttaca 900gcccgtgaat ttgaattagt aagtaaacta aatctccatt ttcacaacct tctcactcag 960ttatttcatc tcctcatgga tagctaccta aacctaaagt tatgataaca atacctgtat 1020tttcatccct atgttacagt tgatacaggt ttcatgaaat actgtgtata ctcaaaagta 1080ctttaaaatt aagccttatg ttgaatagct tatgtagcat acacttctgg catttaaata 1140ttttcatatt gctaactaaa taacgtgttt ctttgagtcc ttacgtttta tacgtttgga 1200gttatctttc agaggtgggc acacaggttt cacccgtagg gtttgggggg cacactcatc 1260ctaaagcctg gtccagagca ttggcacagg ttcctgagac aagagctgtg gttagggagc 1320ttttctgagg atgttcacag gtttattcta aatctagggc aacatcatgt tctcatcccc 1380tctgtaggaa ccaggagcct ggaggcattg ggctctcctt tggactcttc ttcgtctctg 1440ctacaggacg tgtctactca ggcatgtctg tctccctagt tccttatgct ggtccagtga 1500aacacaaaat agacttatat ccctgttcaa actagcacac aaccagcttc tcctgtcaga 1560caaggtgcgc atatgttcac aagcacacac aaacagacta gaaacttagg ggttattatt 1620gggatgtggg gtacatgcac ggggacttct aaaaagaaaa taaattcaaa atagcctccg 1680gcactttgtt tttaaagact cttgctggca gtgtgagtgt aatcctccta tccccccatg 1740gctggtccaa cccagcttca tgtgatcacc tctccctccc tccacacagg gctgggtccc 1800caggatatat aaatgtcttt ggacttcagg cttgagccag cagggccacc catccagaca 1860ccttgcagga gaactttcca gaagaaacct cacccagcct ccacactgct gtccttctct 1920gcacgctgct gcagctgtgg tcccaagatg ccagctctcc atctgctgtt tctggcctgc 1980ttggtgtggg gaatgggggc caggacagca cagttccgaa aggccaatga tcggagtggc 2040cgatgccaat acaccttcac tgtggccagc cccaatgaat ctagctgccc aagggaggac 2100caggccatgt cagccatcca agaccttcag agagacagca gcatccagca tgcagaccta 2160gagtccacca aggcccgggt cagatccctg gagagtctcc tccaccagat gaccttgggc 2220cgagttactg ggacccagga ggcccaagag gggctgcagg gccagttggg tgccctgagg 2280agagaacggg accagctgga gacccaaacc agggatctgg aggcagccta taacaatctc 2340cttcgagata agtcggcttt agaggaagag aagaggcagc tggaacaaga gaatgaagat 2400ttggccagga ggctagaaag cagcagcgag gaggtaacaa ggctgcggag gggccagtgt 2460ccttccaccc agtacccctc tcaggacatg ctgccaggct ccagggaagg taagagtgca 2520gggtggagtg gccacctgac ccagaaggta gcaagtttgc tggtgaccca ttacaggacc 2580cccaggcttc tccttctgtt ttgtcttttc tctcagaaac tgcaaatcca gcatgcagta 2640gtttcattaa ggagagcaaa gcaaacactt ttgcatgctt ctagaaagtt ggctccttgt 2700ttaggtcagt ggatctgagc tcttgtgccc agtcatgaca aaatgatcat ggcccacagc 2760caaatgacaa acatggggcc aggtggcaga tacatatgat 2800 5 680 DNA ArtificialSequence Description of Artificial Sequence; Note = synthetic construct5 aagcttttta attatgccaa tttctccccg attgagacca tcaccctagt tccaatgagc 60taccaacgtg gttcagtcat gttacatctt cagataacaa gtatttggga acatatcaaa 120catcaccctc cacagagtcc gttcttgtgc cctttctact acaagtgcca attttttctc 180tctttgaata cagtctctca gtggaatttg gacacgttgg ccttccagga attgaagtca 240gagttaactg aggttcctgc ttcccaaatc ttgaaggaaa atccatctgg ccgacccagg 300agcaaagaag gagacaaagg tatgaagtta gacttctccc ttttgagcct acctggcctc 360ctctccctct ctccctctct ccctctctcc ctctctccct ctctccctct ctccctctct 420ccctctctcc cctctcccct ccccctctcc ctccctgtgt gtgtgtgtga gtgcatgtat 480atgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgcat gtgcgtgtgc atgtatacct 540tgttctgtgt tcagttcgga aagagcaact gttcacccag aagagaagac aggtgattcc 600ccaaggcaga gttggggaga aggaagctga aacctgtctg ctgccttttc tagacatatg 660tactggaagc caaccttgga 680 6 1456 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 6 ctttgtctat caaggaaaagagcatttgtg cctcaaaaaa aaaaaaaaaa aaaagtgttc 60 gatagaaata tggctgctgtttccagaaaa taacattgac tgttttatta gcaatccctg 120 ctaacactga agtctatgtagaggctaaca cggaagggta tgttgagggg atccgacacc 180 ctcacacaga catacatgcaggcaaaacac caatgcacac aaaagaaaaa caaatgagaa 240 agtcaaggct cacagagctaagtacctcac tggtcacatg gtcagtgggc agcggggttc 300 agaggtcaac ccactctgtctctgccttct ctgttttgcc actactgtcc agtctgcagt 360 ctgtattcgg aagacatagatactaaatac atggcaactc ttttttttgt ttgttttaat 420 tcatcaggat gtggagcgctagtctgggta ggagagccag tcaccctgag gacagctgaa 480 acaatcgctg gcaagtatggagtgtggatg agagacccca agcccaccca cccctacacc 540 caggaaagca catggaggattgacacggtt ggcacagaga tccgccaggt gtttgagtac 600 agtcagataa gccagttcgagcagggctat ccttccaagg tccatgtgct ccctcgggca 660 ctggagagca cgggtgctgtggtgtatgcg gggagcctct atttccaggg ggctgagtcc 720 agaactgtgg tcaggtatgagctagacacg gagaccgtga aggcagagaa ggaaattcct 780 ggagctggct accacggacacttcccgtac gcgtggggtg gctacacaga cattgactta 840 gctgtggatg agagcggcctctgggtcatc tacagcacgg aggaagccaa gggggccata 900 gtcctctcca aattgaacccagcgaacctg gaacttgagc gtacctggga gactaacatc 960 cgtaagcagt ctgtggccaatgcctttgtt atctgtggca tcttgtacac ggtgagcagc 1020 tactcttcag cccatgcaaccgtcaacttc gcctacgaca ctaaaacggg gaccagtaag 1080 accctgacca tcccattcacgaatcgctac aagtacagca gtatgattga ctacaacccc 1140 ctggagagga agctgtttgcctgggacaac ttcaacatgg tcacctatga tatcaagctc 1200 ttggagatgt gaggagcctctatgcctacc agcaaaggcc agaaaaggtg aagttccggg 1260 ctcccgggtg aagcagctgtcagcagaggc agccagatgc atggagtttc tcctcctgct 1320 aaagattttg tttatccgggtcaatgtaca gctagctccc ctctgactga cacgtcctcc 1380 aggcttgtat agtcgcatagactctgttct cttctgtcag ctttcaaagg gctgttcctc 1440 ttttaaaaat cacata 14567 1515 DNA Artificial Sequence Description of Artificial Sequence; Note= synthetic construct 7 atg agg ttc ttc tgt gca cgt tgc tgc agc ttt gggcct gag atg cca 48 Met Arg Phe Phe Cys Ala Arg Cys Cys Ser Phe Gly ProGlu Met Pro 1 5 10 15 gct gtc cag ctg ctg ctt ctg gcc tgc ctg gtg tgggat gtg ggg gcc 96 Ala Val Gln Leu Leu Leu Leu Ala Cys Leu Val Trp AspVal Gly Ala 20 25 30 agg aca gct cag ctc agg aag gcc aat gac cag agt ggccga tgc cag 144 Arg Thr Ala Gln Leu Arg Lys Ala Asn Asp Gln Ser Gly ArgCys Gln 35 40 45 tat acc ttc agt gtg gcc agt ccc aat gaa tcc agc tgc ccagag cag 192 Tyr Thr Phe Ser Val Ala Ser Pro Asn Glu Ser Ser Cys Pro GluGln 50 55 60 agc cag gcc atg tca gtc atc cat aac tta cag aga gac agc agcacc 240 Ser Gln Ala Met Ser Val Ile His Asn Leu Gln Arg Asp Ser Ser Thr65 70 75 80 caa cgc tta gac ctg gag gcc acc aaa gct cga ctc agc tcc ctggag 288 Gln Arg Leu Asp Leu Glu Ala Thr Lys Ala Arg Leu Ser Ser Leu Glu85 90 95 agc ctc ctc cac caa ttg acc ttg gac cag gct gcc agg ccc cag gag336 Ser Leu Leu His Gln Leu Thr Leu Asp Gln Ala Ala Arg Pro Gln Glu 100105 110 acc cag gag ggg ctg cag agg gag ctg ggc acc ctg agg cgg gag cgg384 Thr Gln Glu Gly Leu Gln Arg Glu Leu Gly Thr Leu Arg Arg Glu Arg 115120 125 gac cag ctg gaa acc caa acc aga gag ttg gag act gcc tac agc aac432 Asp Gln Leu Glu Thr Gln Thr Arg Glu Leu Glu Thr Ala Tyr Ser Asn 130135 140 ctc ctc cga gac aag tca gtt ctg gag gaa gag aag aag cga cta agg480 Leu Leu Arg Asp Lys Ser Val Leu Glu Glu Glu Lys Lys Arg Leu Arg 145150 155 160 caa gaa aat gag aat ctg gcc agg agg ttg gaa agc agc agc caggag 528 Gln Glu Asn Glu Asn Leu Ala Arg Arg Leu Glu Ser Ser Ser Gln Glu165 170 175 gta gca agg ctg aga agg ggc cag tgt ccc cag acc cga gac actgct 576 Val Ala Arg Leu Arg Arg Gly Gln Cys Pro Gln Thr Arg Asp Thr Ala180 185 190 cgg gct gtg cca cca ggc tcc aga gaa gtt tct acg tgg aat ttggac 624 Arg Ala Val Pro Pro Gly Ser Arg Glu Val Ser Thr Trp Asn Leu Asp195 200 205 act ttg gcc ttc cag gaa ctg aag tcc gag cta act gaa gtt cctgct 672 Thr Leu Ala Phe Gln Glu Leu Lys Ser Glu Leu Thr Glu Val Pro Ala210 215 220 tcc cga att ttg aag gag agc cca tct ggc tat ctc agg agt ggagag 720 Ser Arg Ile Leu Lys Glu Ser Pro Ser Gly Tyr Leu Arg Ser Gly Glu225 230 235 240 gga gac acc gga tgt gga gaa cta gtt tgg gta gga gag cctctc acg 768 Gly Asp Thr Gly Cys Gly Glu Leu Val Trp Val Gly Glu Pro LeuThr 245 250 255 ctg aga aca gca gaa aca att act ggc aag tat ggt gtg tggatg cga 816 Leu Arg Thr Ala Glu Thr Ile Thr Gly Lys Tyr Gly Val Trp MetArg 260 265 270 gac ccc aag ccc acc tac ccc tac acc cag gag acc acg tggaga atc 864 Asp Pro Lys Pro Thr Tyr Pro Tyr Thr Gln Glu Thr Thr Trp ArgIle 275 280 285 gac aca gtt ggc acg gat gtc cgc cag gtt ttt gag tat gacctc atc 912 Asp Thr Val Gly Thr Asp Val Arg Gln Val Phe Glu Tyr Asp LeuIle 290 295 300 agc cag ttt atg cag ggc tac cct tct aag gtt cac ata ctgcct agg 960 Ser Gln Phe Met Gln Gly Tyr Pro Ser Lys Val His Ile Leu ProArg 305 310 315 320 cca ctg gaa agc acg ggt gct gtg gtg tac tcg ggg agcctc tat ttc 1008 Pro Leu Glu Ser Thr Gly Ala Val Val Tyr Ser Gly Ser LeuTyr Phe 325 330 335 cag ggc gct gag tcc aga act gtc ata aga tat gag ctgaat acc gag 1056 Gln Gly Ala Glu Ser Arg Thr Val Ile Arg Tyr Glu Leu AsnThr Glu 340 345 350 aca gtg aag gct gag aag gaa atc cct gga gct ggc taccac gga cag 1104 Thr Val Lys Ala Glu Lys Glu Ile Pro Gly Ala Gly Tyr HisGly Gln 355 360 365 ttc ccg tat tct tgg ggt ggc tac acg gac att gac ttggct gtg gat 1152 Phe Pro Tyr Ser Trp Gly Gly Tyr Thr Asp Ile Asp Leu AlaVal Asp 370 375 380 gaa gca ggc ctc tgg gtc att tac agc acc gat gag gccaaa ggt gcc 1200 Glu Ala Gly Leu Trp Val Ile Tyr Ser Thr Asp Glu Ala LysGly Ala 385 390 395 400 att gtc ctc tcc aaa ctg aac cca gag aat ctg gaactc gaa caa acc 1248 Ile Val Leu Ser Lys Leu Asn Pro Glu Asn Leu Glu LeuGlu Gln Thr 405 410 415 tgg gag aca aac atc cgt aag cag tca gtc gcc aatgcc ttc atc atc 1296 Trp Glu Thr Asn Ile Arg Lys Gln Ser Val Ala Asn AlaPhe Ile Ile 420 425 430 tgt ggc acc ttg tac acc gtc agc agc tac acc tcagca gat gct acc 1344 Cys Gly Thr Leu Tyr Thr Val Ser Ser Tyr Thr Ser AlaAsp Ala Thr 435 440 445 gtc aac ttt gct tat gac aca ggc aca ggt atc agcaag acc ctg acc 1392 Val Asn Phe Ala Tyr Asp Thr Gly Thr Gly Ile Ser LysThr Leu Thr 450 455 460 atc cca ttc aag aac cgc tat aag tac agc agc atgatt gac tac aac 1440 Ile Pro Phe Lys Asn Arg Tyr Lys Tyr Ser Ser Met IleAsp Tyr Asn 465 470 475 480 ccc ctg gag aag aag ctc ttt gcc tgg gac aacttg aac atg gtc act 1488 Pro Leu Glu Lys Lys Leu Phe Ala Trp Asp Asn LeuAsn Met Val Thr 485 490 495 tat gac atc aag ctc tcc aag atg tga 1515 TyrAsp Ile Lys Leu Ser Lys Met 500 8 504 PRT Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 8 Met ArgPhe Phe Cys Ala Arg Cys Cys Ser Phe Gly Pro Glu Met Pro 1 5 10 15 AlaVal Gln Leu Leu Leu Leu Ala Cys Leu Val Trp Asp Val Gly Ala 20 25 30 ArgThr Ala Gln Leu Arg Lys Ala Asn Asp Gln Ser Gly Arg Cys Gln 35 40 45 TyrThr Phe Ser Val Ala Ser Pro Asn Glu Ser Ser Cys Pro Glu Gln 50 55 60 SerGln Ala Met Ser Val Ile His Asn Leu Gln Arg Asp Ser Ser Thr 65 70 75 80Gln Arg Leu Asp Leu Glu Ala Thr Lys Ala Arg Leu Ser Ser Leu Glu 85 90 95Ser Leu Leu His Gln Leu Thr Leu Asp Gln Ala Ala Arg Pro Gln Glu 100 105110 Thr Gln Glu Gly Leu Gln Arg Glu Leu Gly Thr Leu Arg Arg Glu Arg 115120 125 Asp Gln Leu Glu Thr Gln Thr Arg Glu Leu Glu Thr Ala Tyr Ser Asn130 135 140 Leu Leu Arg Asp Lys Ser Val Leu Glu Glu Glu Lys Lys Arg LeuArg 145 150 155 160 Gln Glu Asn Glu Asn Leu Ala Arg Arg Leu Glu Ser SerSer Gln Glu 165 170 175 Val Ala Arg Leu Arg Arg Gly Gln Cys Pro Gln ThrArg Asp Thr Ala 180 185 190 Arg Ala Val Pro Pro Gly Ser Arg Glu Val SerThr Trp Asn Leu Asp 195 200 205 Thr Leu Ala Phe Gln Glu Leu Lys Ser GluLeu Thr Glu Val Pro Ala 210 215 220 Ser Arg Ile Leu Lys Glu Ser Pro SerGly Tyr Leu Arg Ser Gly Glu 225 230 235 240 Gly Asp Thr Gly Cys Gly GluLeu Val Trp Val Gly Glu Pro Leu Thr 245 250 255 Leu Arg Thr Ala Glu ThrIle Thr Gly Lys Tyr Gly Val Trp Met Arg 260 265 270 Asp Pro Lys Pro ThrTyr Pro Tyr Thr Gln Glu Thr Thr Trp Arg Ile 275 280 285 Asp Thr Val GlyThr Asp Val Arg Gln Val Phe Glu Tyr Asp Leu Ile 290 295 300 Ser Gln PheMet Gln Gly Tyr Pro Ser Lys Val His Ile Leu Pro Arg 305 310 315 320 ProLeu Glu Ser Thr Gly Ala Val Val Tyr Ser Gly Ser Leu Tyr Phe 325 330 335Gln Gly Ala Glu Ser Arg Thr Val Ile Arg Tyr Glu Leu Asn Thr Glu 340 345350 Thr Val Lys Ala Glu Lys Glu Ile Pro Gly Ala Gly Tyr His Gly Gln 355360 365 Phe Pro Tyr Ser Trp Gly Gly Tyr Thr Asp Ile Asp Leu Ala Val Asp370 375 380 Glu Ala Gly Leu Trp Val Ile Tyr Ser Thr Asp Glu Ala Lys GlyAla 385 390 395 400 Ile Val Leu Ser Lys Leu Asn Pro Glu Asn Leu Glu LeuGlu Gln Thr 405 410 415 Trp Glu Thr Asn Ile Arg Lys Gln Ser Val Ala AsnAla Phe Ile Ile 420 425 430 Cys Gly Thr Leu Tyr Thr Val Ser Ser Tyr ThrSer Ala Asp Ala Thr 435 440 445 Val Asn Phe Ala Tyr Asp Thr Gly Thr GlyIle Ser Lys Thr Leu Thr 450 455 460 Ile Pro Phe Lys Asn Arg Tyr Lys TyrSer Ser Met Ile Asp Tyr Asn 465 470 475 480 Pro Leu Glu Lys Lys Leu PheAla Trp Asp Asn Leu Asn Met Val Thr 485 490 495 Tyr Asp Ile Lys Leu SerLys Met 500 9 1473 DNA Artificial Sequence Description of ArtificialSequence; Note = synthetic construct 9 atg cca gct ctc cat ctg ctg tttctg gcc tgc ttg gtg tgg gga atg 48 Met Pro Ala Leu His Leu Leu Phe LeuAla Cys Leu Val Trp Gly Met 1 5 10 15 ggg gcc agg aca gca cag ttc cgaaag gcc aat gat cgg agt ggc cga 96 Gly Ala Arg Thr Ala Gln Phe Arg LysAla Asn Asp Arg Ser Gly Arg 20 25 30 tgc caa tac acc ttc act gtg gcc agcccc aat gaa tct agc tgc cca 144 Cys Gln Tyr Thr Phe Thr Val Ala Ser ProAsn Glu Ser Ser Cys Pro 35 40 45 agg gag gac cag gcc atg tca gcc atc caagac ctt cag aga gac agc 192 Arg Glu Asp Gln Ala Met Ser Ala Ile Gln AspLeu Gln Arg Asp Ser 50 55 60 agc atc cag cat gca gac cta gag tcc acc aaggcc cgg gtc aga tcc 240 Ser Ile Gln His Ala Asp Leu Glu Ser Thr Lys AlaArg Val Arg Ser 65 70 75 80 ctg gag agt ctc ctc cac cag atg acc ttg ggccga gtt act ggg acc 288 Leu Glu Ser Leu Leu His Gln Met Thr Leu Gly ArgVal Thr Gly Thr 85 90 95 cag gag gcc caa gag ggg ctg cag ggc cag ttg ggtgcc ctg agg aga 336 Gln Glu Ala Gln Glu Gly Leu Gln Gly Gln Leu Gly AlaLeu Arg Arg 100 105 110 gaa cgg gac cag ctg gag acc caa acc agg gat ctggag gca gcc tat 384 Glu Arg Asp Gln Leu Glu Thr Gln Thr Arg Asp Leu GluAla Ala Tyr 115 120 125 aac aat ctc ctt cga gat aag tcg gct tta gag gaagag aag agg cag 432 Asn Asn Leu Leu Arg Asp Lys Ser Ala Leu Glu Glu GluLys Arg Gln 130 135 140 ctg gaa caa gag aat gaa gat ttg gcc agg agg ctagaa agc agc agc 480 Leu Glu Gln Glu Asn Glu Asp Leu Ala Arg Arg Leu GluSer Ser Ser 145 150 155 160 gag gag gta aca agg ctg cgg agg ggc cag tgtcct tcc acc cag tac 528 Glu Glu Val Thr Arg Leu Arg Arg Gly Gln Cys ProSer Thr Gln Tyr 165 170 175 ccc tct cag gac atg ctg cca ggc tcc agg gaagtc tct cag tgg aat 576 Pro Ser Gln Asp Met Leu Pro Gly Ser Arg Glu ValSer Gln Trp Asn 180 185 190 ttg gac acg ttg gcc ttc cag gaa ttg aag tcagag tta act gag gtt 624 Leu Asp Thr Leu Ala Phe Gln Glu Leu Lys Ser GluLeu Thr Glu Val 195 200 205 cct gct tcc caa atc ttg aag gaa aat cca tctggc cga ccc agg agc 672 Pro Ala Ser Gln Ile Leu Lys Glu Asn Pro Ser GlyArg Pro Arg Ser 210 215 220 aaa gaa gga gac aaa gga tgt gga gcg cta gtctgg gta gga gag cca 720 Lys Glu Gly Asp Lys Gly Cys Gly Ala Leu Val TrpVal Gly Glu Pro 225 230 235 240 gtc acc ctg agg aca gct gaa aca atc gctggc aag tat gga gtg tgg 768 Val Thr Leu Arg Thr Ala Glu Thr Ile Ala GlyLys Tyr Gly Val Trp 245 250 255 atg aga gac ccc aag ccc acc cac ccc tacacc cag gaa agc aca tgg 816 Met Arg Asp Pro Lys Pro Thr His Pro Tyr ThrGln Glu Ser Thr Trp 260 265 270 agg att gac acg gtt ggc aca gag atc cgccag gtg ttt gag tac agt 864 Arg Ile Asp Thr Val Gly Thr Glu Ile Arg GlnVal Phe Glu Tyr Ser 275 280 285 cag ata agc cag ttc gag cag ggc tat ccttcc aag gtc cat gtg ctc 912 Gln Ile Ser Gln Phe Glu Gln Gly Tyr Pro SerLys Val His Val Leu 290 295 300 cct cgg gca ctg gag agc acg ggt gct gtggtg tat gcg ggg agc ctc 960 Pro Arg Ala Leu Glu Ser Thr Gly Ala Val ValTyr Ala Gly Ser Leu 305 310 315 320 tat ttc cag ggg gct gag tcc aga actgtg gtc agg tat gag cta gac 1008 Tyr Phe Gln Gly Ala Glu Ser Arg Thr ValVal Arg Tyr Glu Leu Asp 325 330 335 acg gag acc gtg aag gca gag aag gaaatt cct gga gct ggc tac cac 1056 Thr Glu Thr Val Lys Ala Glu Lys Glu IlePro Gly Ala Gly Tyr His 340 345 350 gga cac ttc ccg tac gcg tgg ggt ggctac aca gac att gac tta gct 1104 Gly His Phe Pro Tyr Ala Trp Gly Gly TyrThr Asp Ile Asp Leu Ala 355 360 365 gtg gat gag agc ggc ctc tgg gtc atctac agc acg gag gaa gcc aag 1152 Val Asp Glu Ser Gly Leu Trp Val Ile TyrSer Thr Glu Glu Ala Lys 370 375 380 ggg gcc ata gtc ctc tcc aaa ttg aaccca gcg aac ctg gaa ctt gag 1200 Gly Ala Ile Val Leu Ser Lys Leu Asn ProAla Asn Leu Glu Leu Glu 385 390 395 400 cgt acc tgg gag act aac atc cgtaag cag tct gtg gcc aat gcc ttt 1248 Arg Thr Trp Glu Thr Asn Ile Arg LysGln Ser Val Ala Asn Ala Phe 405 410 415 gtt atc tgt ggc atc ttg tac acggtg agc agc tac tct tca gcc cat 1296 Val Ile Cys Gly Ile Leu Tyr Thr ValSer Ser Tyr Ser Ser Ala His 420 425 430 gca acc gtc aac ttc gcc tac gacact aaa acg ggg acc agt aag acc 1344 Ala Thr Val Asn Phe Ala Tyr Asp ThrLys Thr Gly Thr Ser Lys Thr 435 440 445 ctg acc atc cca ttc acg aat cgctac aag tac agc agt atg att gac 1392 Leu Thr Ile Pro Phe Thr Asn Arg TyrLys Tyr Ser Ser Met Ile Asp 450 455 460 tac aac ccc ctg gag agg aag ctgttt gcc tgg gac aac ttc aac atg 1440 Tyr Asn Pro Leu Glu Arg Lys Leu PheAla Trp Asp Asn Phe Asn Met 465 470 475 480 gtc acc tat gat atc aag ctcttg gag atg tga 1473 Val Thr Tyr Asp Ile Lys Leu Leu Glu Met 485 490 10490 PRT Artificial Sequence Description of Artificial Sequence; Note =synthetic construct 10 Met Pro Ala Leu His Leu Leu Phe Leu Ala Cys LeuVal Trp Gly Met 1 5 10 15 Gly Ala Arg Thr Ala Gln Phe Arg Lys Ala AsnAsp Arg Ser Gly Arg 20 25 30 Cys Gln Tyr Thr Phe Thr Val Ala Ser Pro AsnGlu Ser Ser Cys Pro 35 40 45 Arg Glu Asp Gln Ala Met Ser Ala Ile Gln AspLeu Gln Arg Asp Ser 50 55 60 Ser Ile Gln His Ala Asp Leu Glu Ser Thr LysAla Arg Val Arg Ser 65 70 75 80 Leu Glu Ser Leu Leu His Gln Met Thr LeuGly Arg Val Thr Gly Thr 85 90 95 Gln Glu Ala Gln Glu Gly Leu Gln Gly GlnLeu Gly Ala Leu Arg Arg 100 105 110 Glu Arg Asp Gln Leu Glu Thr Gln ThrArg Asp Leu Glu Ala Ala Tyr 115 120 125 Asn Asn Leu Leu Arg Asp Lys SerAla Leu Glu Glu Glu Lys Arg Gln 130 135 140 Leu Glu Gln Glu Asn Glu AspLeu Ala Arg Arg Leu Glu Ser Ser Ser 145 150 155 160 Glu Glu Val Thr ArgLeu Arg Arg Gly Gln Cys Pro Ser Thr Gln Tyr 165 170 175 Pro Ser Gln AspMet Leu Pro Gly Ser Arg Glu Val Ser Gln Trp Asn 180 185 190 Leu Asp ThrLeu Ala Phe Gln Glu Leu Lys Ser Glu Leu Thr Glu Val 195 200 205 Pro AlaSer Gln Ile Leu Lys Glu Asn Pro Ser Gly Arg Pro Arg Ser 210 215 220 LysGlu Gly Asp Lys Gly Cys Gly Ala Leu Val Trp Val Gly Glu Pro 225 230 235240 Val Thr Leu Arg Thr Ala Glu Thr Ile Ala Gly Lys Tyr Gly Val Trp 245250 255 Met Arg Asp Pro Lys Pro Thr His Pro Tyr Thr Gln Glu Ser Thr Trp260 265 270 Arg Ile Asp Thr Val Gly Thr Glu Ile Arg Gln Val Phe Glu TyrSer 275 280 285 Gln Ile Ser Gln Phe Glu Gln Gly Tyr Pro Ser Lys Val HisVal Leu 290 295 300 Pro Arg Ala Leu Glu Ser Thr Gly Ala Val Val Tyr AlaGly Ser Leu 305 310 315 320 Tyr Phe Gln Gly Ala Glu Ser Arg Thr Val ValArg Tyr Glu Leu Asp 325 330 335 Thr Glu Thr Val Lys Ala Glu Lys Glu IlePro Gly Ala Gly Tyr His 340 345 350 Gly His Phe Pro Tyr Ala Trp Gly GlyTyr Thr Asp Ile Asp Leu Ala 355 360 365 Val Asp Glu Ser Gly Leu Trp ValIle Tyr Ser Thr Glu Glu Ala Lys 370 375 380 Gly Ala Ile Val Leu Ser LysLeu Asn Pro Ala Asn Leu Glu Leu Glu 385 390 395 400 Arg Thr Trp Glu ThrAsn Ile Arg Lys Gln Ser Val Ala Asn Ala Phe 405 410 415 Val Ile Cys GlyIle Leu Tyr Thr Val Ser Ser Tyr Ser Ser Ala His 420 425 430 Ala Thr ValAsn Phe Ala Tyr Asp Thr Lys Thr Gly Thr Ser Lys Thr 435 440 445 Leu ThrIle Pro Phe Thr Asn Arg Tyr Lys Tyr Ser Ser Met Ile Asp 450 455 460 TyrAsn Pro Leu Glu Arg Lys Leu Phe Ala Trp Asp Asn Phe Asn Met 465 470 475480 Val Thr Tyr Asp Ile Lys Leu Leu Glu Met 485 490 11 29 DNA ArtificialSequence Description of Artificial Sequence; Note = synthetic construct11 aggggctgca gagggagctg ggcaccctg 29 12 20 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 12atactgccta ggccactgga 20 13 19 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 13 caatgtccgt gtagccacc19 14 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 14 gaactcgaac aaacctggga 20 15 22 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 15 catgctgctg tacttatagc gg 22 16 20 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 16ggctggctcc ccagtatata 20 17 18 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 17 acagctggca tctcaggc18 18 18 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 18 acgttgctcc agctttgg 18 19 19 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 19 gatgactgac atggcctgg 19 20 20 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 20agtggccgat gccagtatac 20 21 20 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 21 ctggtccaag gtcaattggt20 22 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 22 aggccatgtc agtcatccat 20 23 20 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 23 tctctggttt gggtttccag 20 24 18 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 24tgaccttgga ccaggctg 18 25 20 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 25 cctggccaga ttctcatttt20 26 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 26 tggaggaaga gaagaagcga 20 27 20 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 27 ctgctgaact cagagtcccc 20 28 21 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 28aacatagtca atccttgggc c 21 29 20 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 29 taaagaccat gtgggcacaa20 30 22 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 30 ttatggatta agtggtgctt cg 22 31 20 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 31 attctccacg tggtctcctg 20 32 20 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 32aagcccacct acccctacac 20 33 21 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 33 aatagaggct ccccgagtaca 21 34 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 34 atactgccta ggccactgga 20 35 19 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 35 caatgtccgt gtagccacc 19 36 19 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 36tggctaccac ggacacttc 19 37 20 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 37 cattggcgac tgactgctta20 38 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 38 gaactcgaac aaacctggga 20 39 22 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 39 catgctgctg tacttatagc gg 22 40 19 DNA Artificial SequenceDescription of Artificial Sequence; Note = synthetic construct 40agcaagaccc tgaccatcc 19 41 20 DNA Artificial Sequence Description ofArtificial Sequence; Note = synthetic construct 41 agcatctcct tctgccattg20 42 20 DNA Artificial Sequence Description of Artificial Sequence;Note = synthetic construct 42 ttccttcagg ttgggagatg 20 43 20 DNAArtificial Sequence Description of Artificial Sequence; Note = syntheticconstruct 43 gagagcacca ggagatggag 20

1. An isolated nucleic acid molecule comprising a nucleic acid molecule or the complement of a nucleic acid molecule set forth in any of SEQ ID Nos. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43:
 2. An isolated nucleic acid molecule comprising a nucleic acid molecule or the complement of a nucleic acid molecule obtained by amplifying a GLC1A gene with a primer pair selected from the group consisting of SEQ ID Nos 16 and 17, SEQ ID Nos 18 and 19, SEQ ID Nos 20 and 21, SEQ ID Nos 22 and 23, SEQ ID Nos 24 and 25, SEQ ID Nos 26 and 27, SEQ ID Nos 28 and 29, SEQ ID Nos 30 and 31, SEQ ID Nos 32 and 33, SEQ ID Nos 34 and 35, SEQ ID Nos 36 and 37, SEQ ID Nos 38 and 39, SEQ ID Nos 40 and 41, SEQ ID Nos 42 and
 43. 3. An isolated nucleic acid molecule of claim 2, which appears within Exon 1 of FIG. 1 or is the complement of a a nucleic acid molecule, which appears within Exon
 1. 4. An isolated nucleic acid molecule of claim 2, which appears within Exon 2 of FIG. 1 or is the complement of a a nucleic acid molecule, which appears within Exon
 2. 5. An isolated nucleic acid molecule of claim 2, which appears within Exon 3 of FIG. 1 or is the complement of a a nucleic acid molecule, which appears within Exon
 3. 6. An isolated nucleic acid of claim 3, wherein the primer pair is comprised of a member selected from the group consisting of: SEQ ID Nos. 16 and 17; SEQ ID Nos. 18 and 19; SEQ ID Nos. 20 and 21; SEQ ID Nos. 22 and 23; SEQ ID Nos. 24 and 25; and SEQ ID Nos. 26 and
 27. 7. An isolated nucleic acid of claim 4, wherein the primer pair is comprised of SEQ ID Nos. 28 and
 29. 8. An isolated nucleic acid of claim 5, wherein the primer pair is comprised of SEQ ID Nos 30 and 31, SEQ ID Nos 32 and 33, SEQ ID Nos 34 and 35, SEQ ID Nos 36 and 37, SEQ ID Nos 38 and 39, SEQ ID Nos 40 and
 41. 9. An isolated nucleic acid of claim 2, which is upstream of the GLC1A gene and is amplified by SEQ ID Nos 42 and
 43. 10. A method for determining whether a subject has or has the potential for developing primary open angle glaucoma, comprising the steps of: a) obtaining a biological sample containing genomic DNA or a complement thereof from a subject; b) performing an amplification on the genomic DNA using a primer pair selected from the group consisting of SEQ ID Nos 16 and 17, SEQ ID Nos 18 and 19, SEQ ID Nos 20 and 21, SEQ ID Nos 22 and 23, SEQ ID Nos 24 and 25, SEQ ID Nos 26 and 27, SEQ ID Nos 28 and 29, SEQ ID Nos 30 and 31, SEQ ID Nos 32 and 33, SEQ ID Nos 34 and 35, SEQ ID Nos 36 and 37, SEQ ID Nos 38 and 39, SEQ ID Nos 40 and 41, SEQ ID Nos 42 and 43, thereby obtaining an amplification product; and c) analyzing the amplification product for the presence of a mutation, wherein the presence of a mutation indicates that the subject has or has the potential for developing primary open angle glaucoma.
 11. A screening method of claim 10, wherein in step c), the amplification product is analyzed using single strand conformation polymorphism (SSCP) analysis.
 12. A screening method of claim 10, wherein in step c), the amplification product is analyzed by sequencing.
 13. A kit for diagnosing a subject as having primary open angle glaucoma comprising: a) a primer pair selected from the group consisting of: SEQ ID Nos 16 and 17, SEQ ID Nos 18 and 19, SEQ ID Nos 20 and 21, SEQ ID Nos 22 and 23, SEQ ID Nos 24 and 25, SEQ ID Nos 26 and 27, SEQ ID Nos 28 and 29, SEQ ID Nos 30 and 31, SEQ ID Nos 32 and 33, SEQ ID Nos 34 and 35, SEQ ID Nos 36 and 37, SEQ ID Nos 38 and 39, SEQ ID Nos 40 and 41, SEQ ID Nos 42 and 43.; and b) instructions for using the primer pair to perform an amplification. 